Team:TU Munich/Notebook/Labjournal

From 2012.igem.org

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(Transformation of BBa_I742111 (Limonenesynthase) into E.coli XL-1 Blue)
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{{Team:TU_Munich/Header}}
{{Team:TU_Munich/Header}}
{{Team:TU_Munich/LabHeader}}
{{Team:TU_Munich/LabHeader}}
 +
{{Team:TU_Munich/ExCol}}
 +
__NOTOC__
<html>
<html>
<body>
<body>
-
         <form id="labselect"><fieldset class="ui-widget ui-widget-content ui-corner-all">
+
         <form id="labselect"><fieldset class="ui-widget ui-widget-content ui-corner-left">
-
         <b>Display:</b>
+
         <b>Display:</b><br>
-
<input class="labcheckbox" type="checkbox" name="category" value="vector_design" id="ui-test1" /><b style="color: rgb(166, 126, 166)">Vector Design</b>
+
<input class="labcheckbox" type="checkbox" name="category" value="vector_design" id="ui-test1" /><b style="color: rgb(166, 126, 166)">Vector Design</b><br>
-
<input class="labcheckbox" type="checkbox" name="category" value="limonene" id="ui-test2" /><b style="color: rgb(116, 183, 112)">Limonene</b>
+
<input class="labcheckbox" type="checkbox" name="category" value="limonene" id="ui-test2" /><b style="color: rgb(116, 183, 112)">Limonene</b><br>
-
<input class="labcheckbox" type="checkbox" name="category" value="coumaryl" id="ui-test3" /><b style="color: rgb(255, 122, 97)">Coumaryl</b>
+
<input class="labcheckbox" type="checkbox" name="category" value="coumaryl" id="ui-test3" /><b style="color: rgb(255, 122, 97)">Xanthohumol</b><br>
-
<input class="labcheckbox" type="checkbox" name="category" value="thaumatin" id="ui-test4" /><b style="color: rgb(202, 85, 85)">Thaumatin</b>
+
<input class="labcheckbox" type="checkbox" name="category" value="thaumatin" id="ui-test4" /><b style="color: rgb(202, 85, 85)">Thaumatin</b><br>
-
<input class="labcheckbox" type="checkbox" name="category" value="caffeine" id="ui-test5" /><b style="color: rgb(211, 254, 113)">Caffeine</b>
+
<input class="labcheckbox" type="checkbox" name="category" value="caffeine" id="ui-test5" /><b style="color:rgb(192, 167, 4)">Caffeine</b><br>
-
<input class="labcheckbox" type="checkbox" name="category" value="promoter" id="ui-test6" /><b style="color: rgb(115, 208, 255)">Promoter</b>
+
<input class="labcheckbox" type="checkbox" name="category" value="constitutive_promoter" id="ui-test6" /><b style="color: rgb(115, 208, 255)">Constitutive promoter</b><br>
-
         <br><i>You can also click on individual experiments to show/hide them</i>
+
<input class="labcheckbox" type="checkbox" name="category" value="light_switchable_promoter" id="ui-test7"
 +
/><b style="color: rgb(0, 32, 96)">Light-switchable promoter</b><br>
 +
        <input class="labcheckbox" type="checkbox" name="category" value="ethanol_inducible_promoter" id="ui-test8"
 +
/><b style="color: rgb(0, 102, 0)">Ethanol-inducible promoter</b><br>
 +
        <input class="labcheckbox" type="checkbox" name="category" value="integration" id="ui-test9" /><b style="color: rgb(222, 77, 185)">Genome integration</b><br>
 +
        <br><a href="#" id="ExAll">Expand All ...</a><br>
 +
        <a href="#" id="ColAll">Collapse All ...</a><br>
 +
 
 +
         <br><i>You can also click on individual experiments to show/hide them</i><br>
 +
        <b>Jump to:</b><br>
 +
        <a href="#Week_1">Week 1</a> 13.6-17.6<br>
 +
        <a href="#Week_2">Week 2</a> 18.6-24.6<br>
 +
        <a href="#Week_3">Week 3</a> 25.6-1.7<br>
 +
        <a href="#Week_4">Week 4</a> 2.7-8.7<br>
 +
        <a href="#Week_5">Week 5</a> 9.7-15.7<br>
 +
        <a href="#Week_6">Week 6</a> 16.7-22.7<br>
 +
        <a href="#Week_7">Week 7</a> 23.7-29.7<br>
 +
        <a href="#Week_8">Week 8</a> 30.7-5.8<br>
 +
        <a href="#Week_9">Week 9</a> 6.8-12.8<br>
 +
        <a href="#Week_10">Week 10</a> 13.8-19.8<br>
 +
        <a href="#Week_11">Week 11</a> 20.8-26.8<br>
 +
        <a href="#Week_12">Week 12</a> 27.8-2.9<br>
 +
        <a href="#Week_13">Week 13</a> 3.9-9.9<br>
 +
        <a href="#Week_14">Week 14</a> 10.9-16.9<br>
 +
        <a href="#Week_15">Week 15</a> 17.9-23.9<br>
 +
        <a href="#Week_16">Week 16</a> 24.9-27.9
         </fieldset>
         </fieldset>
         </form>
         </form>
 +
        <div id="ladder" class="ui-widget ui-widget-content ui-corner-right">
 +
        <b><u>1 kbp GeneRuler:</u></b><br>
 +
        <img src="http://2012.igem.org/wiki/images/e/e3/TUM12_1000bp.jpg"><br>
 +
        <b><u>100 bp GeneRuler:</u></b><br>
 +
        <img src="http://2012.igem.org/wiki/images/d/da/TUM12_100bp.jpg"><br>
 +
        <b><u>PageRuler Plus:</u></b><br>
 +
        <img src="http://2012.igem.org/wiki/images/8/8c/TUM12_250kDa.jpg"><br>
 +
        </div>
 +
<!-- Habe ich wieder mit reingenommen, weil ich find es wirklich nicht schlecht, weil den Template passt nicht immer, z.B. bei präperative Gele nicht -->
</html>
</html>
 +
= Labjournal =
 +
<hr/>
 +
P1-923 and PCR1-73 are the tube numbers for plasmids/PCR products from our [[Team:TU_Munich/Notebook/Inventory|inventory list (most of the descriptions are in german)]]
-
=June=
+
For a shorter summary of what happened each week, see our [[Team:TU_Munich/Notebook/Meetings|meeting protocols]].
-
<div class="month" id="MJune">
+
 
-
==Wednesday, June 13th==
+
<div class="labbook">
 +
 
 +
=Week 1=
 +
<!--
 +
<p class="vector_design">'''Vector Design (4 Experiments):''' </p>
 +
* Exchange of Multiple Cloning Site of pTUM104
 +
 
 +
<p class="limonene">'''Limonene (2 Experiments):''' </p>
 +
* Transformation with limonene plasmids from Prof. Schwab
 +
 
 +
<html><a class="WDetails" href="#Week_1" id="Week_1">Show Details ...</a></html>
 +
-->
 +
<div class="week" id="WWeek_1">
 +
=='''Wednesday, June 13th'''==
<div class="vector_design">
<div class="vector_design">
-
===Exchange of the Multiple Cloning Site of pYES2 ===
+
===Exchange of the Multiple Cloning Site of pTUM104 ===
-
'''Investigator: Saskia, Daniela'''
+
'''Investigator:''' Saskia, Daniela
-
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
+
'''Aim of the experiment:''' Exchange of the Multiple Cloning Site of pTUM104
'''Hybridisation of the primers O1 with O2 and O3 with O4'''
'''Hybridisation of the primers O1 with O2 and O3 with O4'''
Line 82: Line 134:
</div>
</div>
-
==Thursday, June 14th==
+
=='''Thursday, June 14th'''==
<div class="vector_design">
<div class="vector_design">
-
===Exchange of the Multiple Cloning Site of pYES2 ===
+
===Exchange of the Multiple Cloning Site of pTUM104 ===
-
'''Investigator: Saskia, Daniela'''
+
'''Investigator:''' Saskia, Daniela
-
 
+
-
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
+
-
'''Digestion of pYES2 with HindIII and XbaI'''
+
'''Aim of the experiment:''' Exchange of the Multiple Cloning Site of pTUM104
 +
'''Digestion of pTUM104 with HindIII and XbaI'''
{|cellspacing="0" border="1"
{|cellspacing="0" border="1"
Line 118: Line 169:
Incubation: 37 °C, 1.75 h
Incubation: 37 °C, 1.75 h
-
 
'''DNA preparative gel electrophoresis'''
'''DNA preparative gel electrophoresis'''
Line 126: Line 176:
*70 V, 90 min
*70 V, 90 min
 +
[[File:TUM12_pYES2digested.jpg]]
'''Gelextration'''
'''Gelextration'''
Line 141: Line 192:
<div class="limonene">
<div class="limonene">
 +
===Transformation of plasmids from Prof. Schwab in ''E.coli'' XL-1 Blue===
===Transformation of plasmids from Prof. Schwab in ''E.coli'' XL-1 Blue===
-
'''Investigator: Lara, Andrea'''
+
'''Investigator:''' Lara, Andrea
-
Aim of the experiment: Preparation of the plasmids for transformation
+
'''Aim of the experiment:''' Preparation of the plasmids for transformation
'''Overnight cultures of cells with limonenesynthase-plasmid from Prof. Schwab'''
'''Overnight cultures of cells with limonenesynthase-plasmid from Prof. Schwab'''
Line 158: Line 210:
<div class="vector_design">
<div class="vector_design">
-
===Exchange of the Multiple Cloning Site of pYES2===
+
===Exchange of the Multiple Cloning Site of pTUM104===
-
'''Investigator: Saskia, Daniela'''
+
'''Investigator:''' Saskia, Daniela
-
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
+
'''Aim of the experiment:''' Exchange of the Multiple Cloning Site of pTUM104
'''Analytical DNA gel electrophoresis'''
'''Analytical DNA gel electrophoresis'''
Line 172: Line 224:
*band 4: 3 µl O6 + 7 µl TAE-buffer + 1 µl loading dye
*band 4: 3 µl O6 + 7 µl TAE-buffer + 1 µl loading dye
*band 5: 10 µl DNA ladder (100 bp)
*band 5: 10 µl DNA ladder (100 bp)
 +
[[File:TUM12_pYES2_and_primer.jpg]]
'''Ligation of Plasmid P5 (pYES2 digested) with the hybridized primers O5 and O6'''
'''Ligation of Plasmid P5 (pYES2 digested) with the hybridized primers O5 and O6'''
Line 221: Line 274:
'''Transformation of E. coli with ligated products (P6)'''
'''Transformation of E. coli with ligated products (P6)'''
*competent cells: SHXL1 Blue (by Simon)
*competent cells: SHXL1 Blue (by Simon)
-
*Transformation with
+
*Transformation with ligation product (P6) and negative control
-
**ligation product (P6)
+
-
**negative control
+
results:
results:
Line 233: Line 284:
<div class="limonene">
<div class="limonene">
 +
===Transformation of plasmids from Prof. Schwab into ''E.coli'' XL-1 Blue===
===Transformation of plasmids from Prof. Schwab into ''E.coli'' XL-1 Blue===
-
'''Investigator: Andrea'''
+
'''Investigator:''' Andrea
-
Aim of the experiment: Preparation of the plasmids for transformation
+
'''Aim of the experiment:''' Preparation of the plasmids for transformation
'''Determination of the concentration with Nano Drop'''
'''Determination of the concentration with Nano Drop'''
Line 261: Line 313:
<div class="vector_design">
<div class="vector_design">
-
===Exchange of the Multiple Cloning Site of pYES2===
+
===Exchange of the Multiple Cloning Site of pTUM104===
-
'''Investigator: Saskia, Daniela'''
+
'''Investigator:''' Saskia, Daniela
-
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
+
'''Aim of the experiment:''' Exchange of the Multiple Cloning Site of pTUM104
'''Picking clones for Miniprep'''
'''Picking clones for Miniprep'''
Line 272: Line 324:
</div>
</div>
 +
</div>
 +
 +
=Week 2=
 +
<!--
 +
<p class="vector_design">'''Vector Design (4 Experiments):'''</p>
 +
* Exchange of Multiple Cloning Site of pTUM104
 +
* Quick Change mutagenisis of pTUM104 for RFC25 compatibility
 +
 +
<p class="limonene">'''Limonene (3 Experiments):'''</p>
 +
* Transformation with limonene BioBricks
 +
 +
<p class="coumaryl">'''Xanthohumol (2 Experiments):'''</p>
 +
* Amplification of plasmids containing the genes for the enzymes PAL, 4Cl and CHS
 +
 +
<p class="light_switchable_promoter">'''Light Switchable Promoter (1 Experiment):'''</p>
 +
* Transformation with heme oxygenase and LexA BioBricks to them RFC25 compatible later on
 +
 +
<html><a class="WDetails" href="#Week_2" id="Week_2">Show Details ...</a></html>
 +
-->
 +
<div class="week" id="WWeek_2">
== '''Monday, June 18th''' ==
== '''Monday, June 18th''' ==
<div class="vector_design">
<div class="vector_design">
-
===Exchange of the Multiple Cloning Site of pYES2===
+
===Exchange of the Multiple Cloning Site of pTUM104===
-
'''Investigator: Saskia, Daniela'''
+
'''Investigator:''' Saskia, Daniela
-
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
+
'''Aim of the experiment:''' Exchange of the Multiple Cloning Site of pTUM104
'''Miniprep of transformed E.coli with P6'''
'''Miniprep of transformed E.coli with P6'''
*QIAprepS Spin Miniprep Kit
*QIAprepS Spin Miniprep Kit
-
**step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)
+
*step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)
*the 10 Minipreps were named: P7 - P16
*the 10 Minipreps were named: P7 - P16
-
 
'''Determination of the concentration with Nano Drop'''
'''Determination of the concentration with Nano Drop'''
Line 379: Line 450:
'''Analytical gel electrophoresis of P7-P16'''
'''Analytical gel electrophoresis of P7-P16'''
*gel: 1.5 %
*gel: 1.5 %
-
*gel 1:  
+
gel 1:  
-
** band 1: 10 µl DNA ladder (1 kb)
+
*band 1: 10 µl DNA ladder (1 kb)
-
** band 2: 3 µl pYES2 SH 1.7.3 digested with HindIII and XbaI + 7 µl TAE buffer + 1 µl loading dye
+
*band 2: 3 µl pTUM104 SH 1.7.3 digested with HindIII and XbaI + 7 µl TAE buffer + 1 µl loading dye
-
** band 3 - 12: 3 µl P7-P16 digested with HindIII and XbaI + 7 µl TAE buffer + 1 µl loading dye
+
*band 3 - 12: 3 µl P7-P16 digested with HindIII and XbaI + 7 µl TAE buffer + 1 µl loading dye
 +
[[File:TUM12_pYES_new_mcs_digested_with_Xbal_and_HindIII.jpg]]
-
*gel 2:
+
gel 2:
-
** band 1: 10 µl DNA ladder (1 kb)
+
*band 1: 10 µl DNA ladder (1 kb)
-
** band 2: 3 µl pYES2 SH 1.7.3 digested with NgoMIV + 7 µl TAE buffer + 1 µl loading dye
+
*band 2: 3 µl pTUM104 SH 1.7.3 digested with NgoMIV + 7 µl TAE buffer + 1 µl loading dye
-
** band 3 - 12: 3 µl P7-P16 digested with NgoMIV + 7 µl TAE buffer + 1 µl loading dye
+
*band 3 - 12: 3 µl P7-P16 digested with NgoMIV + 7 µl TAE buffer + 1 µl loading dye
 +
[[File:TUM12_pYES_new_mcs_digested_with_NgoMIV.jpg]]
</div>
</div>
Line 395: Line 468:
===Transformation of BBa_I742111 (Limonenesynthase) into ''E.coli'' XL-1 Blue===
===Transformation of BBa_I742111 (Limonenesynthase) into ''E.coli'' XL-1 Blue===
-
'''Investigator: Andrea'''
+
'''Investigator:''' Andrea
-
Aim of the experiment: Transformation
+
'''Aim of the experiment:''' Transformation
* for each Biobrick 100 µl cells were used and pooled together with 2 µl of plasmid DNA
* for each Biobrick 100 µl cells were used and pooled together with 2 µl of plasmid DNA
Line 418: Line 491:
<div class="vector_design">
<div class="vector_design">
-
===Exchange of the Multiple Cloning Site of pYES2===
+
===Exchange of the Multiple Cloning Site of pTUM104===
-
'''Investigator: Saskia, Daniela'''
+
'''Investigator:''' Saskia, Daniela
-
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
+
'''Aim of the experiment:''' Exchange of the Multiple Cloning Site of pTUM104
-
'''Sequencing of P13 and P14: pYES2 with new MCS'''
+
'''Sequencing of P13 and P14: pTUM104 with new MCS'''
-
*sequencing primer:
+
sequencing primer:
-
**1.6 µM forward primer O9
+
*1.6 µM forward primer O9
-
**DNA P13 and P14
+
*DNA P13 and P14
 +
 
 +
The Multiple Cloning Site was exchanged successfully!!!
</div>
</div>
<div class="limonene">
<div class="limonene">
 +
===Transformation of BBa_I742111 (Limonenesynthase) into ''E.coli'' XL-1 Blue===
===Transformation of BBa_I742111 (Limonenesynthase) into ''E.coli'' XL-1 Blue===
-
'''Investigator: Daniela'''
+
'''Investigator:''' Daniela
-
Aim of the experiment: Transformation
+
'''Aim of the experiment:''' Transformation
'''Picking of Clones'''
'''Picking of Clones'''
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===Transformation of BBa_I742111 and plasmids from Prof. Schwab into ''E.coli'' XL-1 Blue===
===Transformation of BBa_I742111 and plasmids from Prof. Schwab into ''E.coli'' XL-1 Blue===
-
'''Investigator: Lara, Andrea'''
+
'''Investigator: Lara, Andrea
-
 
+
-
Aim of the experiment: Controll of Transformation
+
 +
'''Aim of the experiment:''' Controll of Transformation
'''Controll digestion'''
'''Controll digestion'''
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'''Analytic Gelelectrophoresis'''
'''Analytic Gelelectrophoresis'''
 +
 +
[[File:21.06.12.png|400px]]
</div>
</div>
== '''Friday, June 22nd''' ==
== '''Friday, June 22nd''' ==
-
 
<div class="coumaryl">
<div class="coumaryl">
=== Transformation of ''E.coli'' XL1-Blue with pKS2µHyg-PAL-4Cl-CHS===
=== Transformation of ''E.coli'' XL1-Blue with pKS2µHyg-PAL-4Cl-CHS===
-
'''Investigator: Ingmar, Volker'''
+
'''Investigator:''' Ingmar, Volker
-
Aim of the experiment: Plasmid amplification
+
'''Aim of the experiment:''' Plasmid amplification
Operation Sequence:
Operation Sequence:
Line 553: Line 629:
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to remove NgoMIV from pYES2 ===
+
=== Quick Change mutagenis to remove NgoMIV from pTUM104 ===
-
'''Investigator: Ingmar, Volker'''
+
'''Investigator:''' Ingmar, Volker
-
Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.
+
'''Aim of the experiment:''' Generation of an RFC 25 compatible version of the pTUM104 Vector.
'''PCR'''<br>
'''PCR'''<br>
Line 584: Line 660:
|-
|-
|0.5 µl
|0.5 µl
-
|Pfu Turbo DNA polymerase (2.5 U / µl)
+
|Pfu Ultra II DNA polymerase (2.5 U / µl)
|}
|}
Line 633: Line 709:
=== Miniprep of ''E.coli'' XL1-Blue with pKS2µHyg-PAL-4Cl-CHS ===
=== Miniprep of ''E.coli'' XL1-Blue with pKS2µHyg-PAL-4Cl-CHS ===
-
'''Investigator: Ingmar, Volker'''
+
'''Investigator:''' Ingmar, Volker
-
Aim of the experiment: Plasmid purification
+
'''Aim of the experiment:''' Plasmid purification
Operation Sequence:
Operation Sequence:
Line 645: Line 721:
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to remove NgoMIV from pYES2 ===
+
=== Quick Change mutagenis to remove NgoMIV from pTUM104 ===
-
'''Investigator: Ingmar, Volker'''
+
'''Investigator:''' Ingmar, Volker
-
Aim of the experiment: Removal of a NgoMIV restriction site in the backbone of pYes2.
+
'''Aim of the experiment:''' Removal of a NgoMIV restriction site in the backbone of pTUM104.
Operational sequence:
Operational sequence:
Line 655: Line 731:
</div>
</div>
-
<div class="promoter">
+
<div class="light_switchable_promoter">
-
=== Transformation of 2 Biobricks into E. coli XL1-Blue ===
+
-
'''Investigator: Jeffery Truong'''
+
=== Transformation of 2 Biobricks into ''E. coli'' XL1-Blue ===
 +
 
 +
'''Investigator:''' Jeffery Truong
-
Aim of the experiment: Transformation of Biobricks into E. coli for plasmid propagation for PCR with new RFC pre- and suffix primer in order to do protein fusions.
+
'''Aim of the experiment:''' Transformation of Biobricks into ''E. coli'' for plasmid propagation for PCR with new RFC pre- and suffix primer in order to do protein fusions.
* 2 Biobricks from the distribution kit were used: First, LexA (BBa_K105005, Plate 3 Well 9E) in the pSB1A2 plasmid with ampicillin resistance and second, the heme oxygenase (BBa_I15008, Plate 2 Well 13J) in the pSB2K3 plasmid with kanamycin resistance.
* 2 Biobricks from the distribution kit were used: First, LexA (BBa_K105005, Plate 3 Well 9E) in the pSB1A2 plasmid with ampicillin resistance and second, the heme oxygenase (BBa_I15008, Plate 2 Well 13J) in the pSB2K3 plasmid with kanamycin resistance.
Line 685: Line 762:
* These 4 plates were put at 37&nbsp;°C overnight
* These 4 plates were put at 37&nbsp;°C overnight
 +
</div>
</div>
 +
</div>
 +
 +
=Week 3=
 +
<!--
 +
<p class="vector_design">'''Vector Design (9 Experiments):'''</p>
 +
* Quick Change mutagenisis of pTUM104 for RFC25 compatibility and
 +
 +
<p class="limonene">'''Limonene (1 Experiment):'''</p>
 +
* Repetition of analytical gelectrophoresis
 +
 +
<p class="coumaryl">'''Xanthohumol (3 Experiments):'''</p>
 +
* PCR of PAL, 4CL, CHS, OMT
 +
 +
<p class="light_switchable_promoter">'''Light Switchable Promoter (2 Experiments):'''</p>
 +
* Verification of transformations (positive)
 +
<html><a class="WDetails" href="#Week_3" id="Week_3">Show Details ...</a></html>
 +
-->
 +
<div class="week" id="WWeek_3">
== '''Monday, June 25th''' ==
== '''Monday, June 25th''' ==
<div class="vector_design">
<div class="vector_design">
-
=== Miniprep of ''E. coli'' XL1-Blue with pYes2_RFC25 MCS 1.2 ===
+
=== Miniprep of ''E. coli'' XL1-Blue with pTUM104_RFC25 MCS 1.2 ===
-
'''Investigator: Alois, Martin'''
+
'''Investigator:''' Alois, Martin
-
Aim of the experiment: proof of successful removal of NgoMIV in the backbone of pYes2
+
'''Aim of the experiment:''' proof of successful removal of NgoMIV in the backbone of pTUM104
Operation Sequence:
Operation Sequence:
-
* Mini prep of pYes2 1.2. The resulting purified DNA is P33.
+
* Mini prep of pTUM104 1.2. The resulting purified DNA is P33.
-
* Control digest of pYes2_RFC25 MCS 1.2 and p13 (+ analytical gel electrophoresis: 90 V, 1 h:
+
* Control digest of pTUM104_RFC25 MCS 1.2 and p13 (+ analytical gel electrophoresis: 90 V, 1 h:
  *  15 µl ddH20
  *  15 µl ddH20
  *  2 µl NEBuffer4
  *  2 µl NEBuffer4
  * 0,5 µl NgoMIV
  * 0,5 µl NgoMIV
-
  * 2,5 µl pYes2 1.2/p13
+
  * 2,5 µl pTUM104 1.2/p13
  * 37°C, 1 h.
  * 37°C, 1 h.
</div>
</div>
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to remove SpeI from pYES2_RFC25 MCS 1.2 ===
+
=== Quick Change mutagenis to remove SpeI from pTUM104_RFC25 MCS 1.2 ===
-
'''Investigator: Ingmar, Volker'''
+
'''Investigator:''' Ingmar, Volker
-
Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.
+
'''Aim of the experiment:''' Generation of an RFC 25 compatible version of the pTUM104 Vector.
'''PCR'''<br>
'''PCR'''<br>
Line 738: Line 834:
|-
|-
|0.5 µl
|0.5 µl
-
|Pfu Turbo DNA polymerase (2.5 U / µl)
+
|Pfu Ultra II DNA polymerase (2.5 U / µl)
|}
|}
Line 770: Line 866:
|}
|}
*The procedure was furthermore applied to P13 and P14.
*The procedure was furthermore applied to P13 and P14.
-
*The vector resulting from the '''PCR-product was named pYes2_RFC25 MCS 1.3'''.
+
*The vector resulting from the '''PCR-product was named pTUM104_RFC25 MCS 1.3'''.
*Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
*Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
Line 785: Line 881:
</div>
</div>
-
<div class="promoter">
+
<div class="light_switchable_promoter">
 +
 
=== Picking of E.&nbsp;coli cells on antibiotic selection plates: pSB1A2 plasmid with BBa_K105005 (LexA) and pSB2K3 plasmid BBa_I15008 (heme oxygenase) ===
=== Picking of E.&nbsp;coli cells on antibiotic selection plates: pSB1A2 plasmid with BBa_K105005 (LexA) and pSB2K3 plasmid BBa_I15008 (heme oxygenase) ===
-
'''Investigator: Jeffery Truong'''
+
'''Investigator:''' Jeffery Truong
-
Aim of the experiment: Picking colonies from transformed E.&nbsp;coli XL1-Blue, 4x picked for each Biobrick.
+
'''Aim of the experiment:''' Picking colonies from transformed E.&nbsp;coli XL1-Blue, 4x picked for each Biobrick.
* pSB1A2 plasmid with BBa_K105005 (LexA): Colonies were on both ampicillin selection plates, the one with diluted cell suspension and the one with concentrated E.&nbsp;coli cell suspension. Typical E.&nbsp;coli colony morphology. Picking was performed on the plate with diluted cell suspension.
* pSB1A2 plasmid with BBa_K105005 (LexA): Colonies were on both ampicillin selection plates, the one with diluted cell suspension and the one with concentrated E.&nbsp;coli cell suspension. Typical E.&nbsp;coli colony morphology. Picking was performed on the plate with diluted cell suspension.
Line 801: Line 898:
* These tubes were transferred in a cell culture shaker at 37&nbsp;°C and were incubated overnight
* These tubes were transferred in a cell culture shaker at 37&nbsp;°C and were incubated overnight
 +
</div>
</div>
Line 806: Line 904:
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to remove SpeI from  pYes2_RFC25 MCS ===
+
=== Quick Change mutagenis to remove SpeI from  pTUM104_RFC25 MCS ===
-
'''Investigator: Ingmar'''
+
'''Investigator:''' Ingmar
-
Aim of the experiment: Removal of a SpeI restriction site in the backbone of pYes2.
+
'''Aim of the experiment:''' Removal of a SpeI restriction site in the backbone of pTUM104.
Operational sequence:
Operational sequence:
Line 880: Line 978:
=== Verification of the PCR products P30, P31 and P33===
=== Verification of the PCR products P30, P31 and P33===
-
'''Investigator: Saskia, Jara'''
+
'''Investigator:''' Saskia, Jara
-
Aim of the experiment: Verification of the PCR produts P30, P31 and P33
+
'''Aim of the experiment:''' Verification of the PCR produts P30, P31 and P33
'''Nano Drop'''
'''Nano Drop'''
Line 928: Line 1,026:
<div class="coumaryl">
<div class="coumaryl">
-
=== PCR of PAL, 4CL, CHS, OMT (Coumaryl-CoA) ===
+
=== PCR of PAL, 4CL, CHS, OMT (Xanthohumol-CoA) ===
'''Investigator: Daniela, Mary'''
'''Investigator: Daniela, Mary'''
Line 957: Line 1,055:
| 4CL + || 4CL || +|| O21 and O20
| 4CL + || 4CL || +|| O21 and O20
|}
|}
-
 
'''Reaction batch'''
'''Reaction batch'''
Line 1,019: Line 1,116:
|-
|-
|}
|}
-
 
PCR purification
PCR purification
*Purification was done using QIAquick PCR Purification Kit (250)
*Purification was done using QIAquick PCR Purification Kit (250)
-
 
Analytical Gel Electrophoresis:
Analytical Gel Electrophoresis:
Line 1,032: Line 1,127:
</div>
</div>
-
<div class="promoter">
+
<div class="light_switchable_promoter">
=== Miniprep and analytical gel of picked transformed overnight culture with pSB1A2 plasmid with BBa_K105005 (LexA) pSB2K3 plasmid BBa_I15008 (heme oxygenase) ===
=== Miniprep and analytical gel of picked transformed overnight culture with pSB1A2 plasmid with BBa_K105005 (LexA) pSB2K3 plasmid BBa_I15008 (heme oxygenase) ===
Line 1,079: Line 1,174:
* Analytical Gel okay, but samples interchanged
* Analytical Gel okay, but samples interchanged
[[File:TUM12_Gel1editedit.jpg|500px|Analytical gel after digestion with XbaI and PstI]]
[[File:TUM12_Gel1editedit.jpg|500px|Analytical gel after digestion with XbaI and PstI]]
 +
 +
</div>
 +
 +
== '''Wednesday, June 27th''' ==
 +
 +
<div class="limonene">
 +
=== Repetition of analytic gel of 21st June===
 +
 +
'''Investigator: Andrea, Lara'''
 +
 +
Buffer systems were adjusted. -> only use of one buffer per reaction.
 +
 +
[[File:27.06.12.png|400px]]
 +
</div>
</div>
Line 1,146: Line 1,255:
'''Investigator: Katrin, Mary'''
'''Investigator: Katrin, Mary'''
-
 
-
picture follows!
 
Gelextraction of 4CL+, 4CL-, CHS+, CHS- (bands are as expected; +=with consensus-sequence, -=without consensus-sequence)
Gelextraction of 4CL+, 4CL-, CHS+, CHS- (bands are as expected; +=with consensus-sequence, -=without consensus-sequence)
 +
 +
[[File:29.06. prepgel4CL.jpg|500px|preparative gel of 4CL]]
 +
 +
[[File:29.06. prepgelCHS.jpg|500px|preparative gel of CHS]]
DNA-purification with Kit from Quiagen  
DNA-purification with Kit from Quiagen  
Line 1,155: Line 1,266:
</div>
</div>
<div class="vector_design">
<div class="vector_design">
-
===  Quick Change mutagenesis to remove PstI in URA3 from pYES2_RFC25 MCS 1.2 ===
+
 
 +
===  Quick Change mutagenesis to remove PstI in URA3 from pTUM104_RFC25 MCS 1.2 ===
'''Investigator: Ingmar'''
'''Investigator: Ingmar'''
-
Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.
+
Aim of the experiment: Generation of an RFC 25 compatible version of the pTUM104 Vector.
'''PCR'''<br>
'''PCR'''<br>
Line 1,234: Line 1,346:
== '''Saturday, June 30th''' ==
== '''Saturday, June 30th''' ==
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to remove PstI in the URA 3 gene from  pYes2_RFC25 MCS ===
+
=== Quick Change mutagenis to remove PstI in the URA 3 gene from  pTUM104_RFC25 MCS ===
'''Investigator: Ingmar'''
'''Investigator: Ingmar'''
-
Aim of the experiment: Removal of a PstI restriction site in the backbone of pYes2.
+
Aim of the experiment: Removal of a PstI restriction site in the backbone of pTUM104.
Operational sequence:
Operational sequence:
* For each transformation of the PCR-products of P29 and P30 a single clone was picked an transferred to 6 ml LB Amp. Incubation overnight at 37°C 180 rpm. The transfomation with the PCR product of P31, P32 and P33 was not successfull. Therfore no clone could be picked from these plates and four instead of one clone was picked from the plates containing the  transformations of P29.
* For each transformation of the PCR-products of P29 and P30 a single clone was picked an transferred to 6 ml LB Amp. Incubation overnight at 37°C 180 rpm. The transfomation with the PCR product of P31, P32 and P33 was not successfull. Therfore no clone could be picked from these plates and four instead of one clone was picked from the plates containing the  transformations of P29.
-
</div>
 
</div>
</div>
-
=July=
 
-
<div class="month" id="MJuly">
 
== '''Sunday, July 1st''' ==
== '''Sunday, July 1st''' ==
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to remove PstI in the URA 3 gene from  pYes2_RFC25 MCS ===
+
=== Quick Change mutagenis to remove PstI in the URA 3 gene from  pTUM104_RFC25 MCS ===
'''Investigator: Ingmar'''
'''Investigator: Ingmar'''
-
Aim of the experiment: Removal of a PstI restriction site in the backbone of pYes2.
+
Aim of the experiment: Removal of a PstI restriction site in the backbone of pTUM104.
Operational sequence:
Operational sequence:
Line 1,325: Line 1,434:
<div class="vector_design">
<div class="vector_design">
-
===  Quick Change mutagenesis to remove PstI in the 2µ ori from pYES2_RFC25 MCS  ===
+
===  Quick Change mutagenesis to remove PstI in the 2µ ori from pTUM104_RFC25 MCS  ===
'''Investigator: Ingmar'''
'''Investigator: Ingmar'''
-
Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.
+
Aim of the experiment: Generation of an RFC 25 compatible version of the pTUM104 Vector.
'''PCR'''<br>
'''PCR'''<br>
Line 1,353: Line 1,462:
|-
|-
|0.5 µl
|0.5 µl
-
|Pfu Turbo DNA polymerase (2.5 U / µl)
+
|Pfu Ultra II DNA polymerase (2.5 U / µl)
|}
|}
Line 1,377: Line 1,486:
|-
|-
|0.5 µl
|0.5 µl
-
|Pfu Turbo DNA polymerase (2.5 U / µl)
+
|Pfu Ultra II DNA polymerase (2.5 U / µl)
|}
|}
-
 
'''PCR cycling parameters'''
'''PCR cycling parameters'''
Line 1,423: Line 1,531:
</div>
</div>
 +
</div>
 +
 +
=Week 4=
 +
<!--
 +
<p class="vector_design">'''Vector Design (6 Experiments):'''</p>
 +
* Further Quickchanges for RFC25 compatibility and insertion of Ala before the strep tag
 +
 +
<p class="limonene">'''Limonene (8 Experiments):'''</p>
 +
* Transformation with Schwab plasmids
 +
* PCR of both Schwab and BioBrick to make them RFC25 compatible
 +
 +
<p class="coumaryl">'''Xanthohumol (4 Experiments):'''</p>
 +
* Repetition of PCR with PAL and troubleshooting
 +
 +
<p class="thaumatin">'''Thaumatin (2 Experiments):'''</p>
 +
* Miniprepping reporter BioBricks gfp/egfp/yfp
 +
 +
<p class="constitutive_promoter">'''Constitutive Promoter (1 Experiment):'''</p>
 +
* Transformation with BioBricks
 +
 +
<p class="light_switchable_promoter">'''Light Switchable Promoter (2 Experiments):'''</p>
 +
* PCR of LexA BioBricks to introduce RFC25
 +
 +
<html><a class="WDetails" href="#Week_4" id="Week_4">Show Details ...</a></html>
 +
-->
 +
<div class="week" id="WWeek_4">
== '''Monday, July 2nd''' ==
== '''Monday, July 2nd''' ==
<div class="coumaryl">
<div class="coumaryl">
Line 1,456: Line 1,590:
|bidest. sterile Water
|bidest. sterile Water
|}
|}
-
 
'''PCR cycling parameters'''
'''PCR cycling parameters'''
Line 1,492: Line 1,625:
|}
|}
</div>
</div>
-
<div class="promoter">
+
<div class="light_switchable_promoter">
=== PCR of LexA with primers including the RFC25 pre- and suffix ===
=== PCR of LexA with primers including the RFC25 pre- and suffix ===
Line 1,562: Line 1,695:
== '''Tuesday, July 3rd''' ==
== '''Tuesday, July 3rd''' ==
-
<div class="promoter">
+
<div class="light_switchable_promoter">
=== Analytic gelelectrophoresis of cleaned up PCR product from LexA with primer containing RFC25 pre- and suffix ===
=== Analytic gelelectrophoresis of cleaned up PCR product from LexA with primer containing RFC25 pre- and suffix ===
Line 1,589: Line 1,722:
===LS: Plating of Schwab expression stains #106 & #108 which contain lavendula limonene synthase===
===LS: Plating of Schwab expression stains #106 & #108 which contain lavendula limonene synthase===
-
'''Investigator: Lara Kuntz'''
+
'''Investigator: Lara'''
Aim of the experiment: To get colonies of BL21 strains containing lavendula LS for amplification and subsequent plasmid extraction.
Aim of the experiment: To get colonies of BL21 strains containing lavendula LS for amplification and subsequent plasmid extraction.
Line 1,597: Line 1,730:
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to remove PstI in the 2µ Ori from  pYes2_RFC25 MCS ===
+
 
 +
=== Quick Change mutagenis to remove PstI in the 2µ Ori from  pTUM104_RFC25 MCS ===
'''Investigator: Ingmar'''
'''Investigator: Ingmar'''
-
Aim of the experiment: Removal of a PstI restriction site in the backbone of pYes2.
+
Aim of the experiment: Removal of a PstI restriction site in the backbone of pTUM104.
Operational sequence:
Operational sequence:
Line 1,608: Line 1,742:
<div class="limonene">
<div class="limonene">
-
=== PCR of BBa_I742111 (Limonenesynthase) Clone 3 (Trafo 19.06.12) ===
+
=== PCR of BBa_I742111 (Limonenesynthase) Clone 3 (Transformation of 19.06.12) ===
'''Investigator: Andrea'''
'''Investigator: Andrea'''
Line 1,703: Line 1,837:
'''Analytical Gelelectrophoresis'''
'''Analytical Gelelectrophoresis'''
* 5 µl DNA + 1 µl loading buffer
* 5 µl DNA + 1 µl loading buffer
-
</div>
 
 +
[[File:03.07.12.png|400px]]
 +
</div>
== '''Wednesday, July 4th''' ==
== '''Wednesday, July 4th''' ==
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to remove PstI in the 2µ Ori from  pYes2_RFC25 MCS ===
+
=== Quick Change mutagenis to remove PstI in the 2µ Ori from  pTUM104_RFC25 MCS ===
'''Investigator: Ingmar'''
'''Investigator: Ingmar'''
-
Aim of the experiment: Removal of a PstI restriction site in the backbone of pYes2.
+
Aim of the experiment: Removal of a PstI restriction site in the backbone of pTUM104.
Operational sequence:
Operational sequence:
Line 1,760: Line 1,895:
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to  insert Ala in front of the Strep - tag II in pYES2_RFC25 MCS  ===
+
=== Quick Change mutagenis to  insert Ala in front of the Strep - tag II in pTUM104_RFC25 MCS  ===
'''Investigator: Ingmar'''
'''Investigator: Ingmar'''
-
Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector; operating purfication possibility via Strep-tag II.
+
Aim of the experiment: Generation of an RFC 25 compatible version of the pTUM104 Vector; operating purfication possibility via Strep-tag II.
'''PCR'''<br>
'''PCR'''<br>
Line 1,840: Line 1,975:
===Analytical Gelelektrophoresis of PCR-Products of PAL===
===Analytical Gelelektrophoresis of PCR-Products of PAL===
-
 
'''Investigator: Mary'''
'''Investigator: Mary'''
Line 1,849: Line 1,983:
analytical gelelectrophoresis of PAL+, PAL-; expected band at 2,1 kb
analytical gelelectrophoresis of PAL+, PAL-; expected band at 2,1 kb
-
piture follows
+
Analytical Gel Electrophoresis:
 +
[[File:TUM12_20120704_PAL-PCR_v2.tiff|800px]]
-
-> PCR was not successful
+
-> PCR was not successful, no band at 2,1 kb
 +
 
 +
-> next steps: new Design of Primer and repetition of PCR with new primers
</div>
</div>
<div class="limonene">
<div class="limonene">
 +
===Picking of clones of Schwab expression stains #106 & #108===
===Picking of clones of Schwab expression stains #106 & #108===
Line 1,864: Line 2,002:
*2 clones of every stain were picked
*2 clones of every stain were picked
*Incubation at 37 °C in LB + Amp (#108) / LB + Kan (#106)
*Incubation at 37 °C in LB + Amp (#108) / LB + Kan (#106)
-
</div>
 
</div>
</div>
Line 1,946: Line 2,083:
Plasmids were digested, 2 µl loading dye (10x) was added to each sample. Gel was loaded in the following order:
Plasmids were digested, 2 µl loading dye (10x) was added to each sample. Gel was loaded in the following order:
-
1. 6 µl gene ruler 1 kb, 2.-6. 11 µl of: P39, P40, P41, P42, Coumaryl-Plasmid (Kathrin)
+
1. 6 µl gene ruler 1 kb, 2.-6. 11 µl of: P39, P40, P41, P42, Xanthohumol-Plasmid (Katrin)
[[File:TUM12_VerdauSchwabPlasmide0507.png]]
[[File:TUM12_VerdauSchwabPlasmide0507.png]]
Line 1,977: Line 2,114:
analytical gelelectrophoresis: expected band at 3,14 kb (Gal-PAL-XK)
analytical gelelectrophoresis: expected band at 3,14 kb (Gal-PAL-XK)
-
picture follows
+
[[File:Analytical agarosegel pKS2µHyg-PAL-4Cl-CHS.png|500px|Analytical gelelectrophoresis of PCR product of PAL]]
-> experiment was successful, PAL is part of the plasmid pKS2µHyg-PAL-4Cl-CHS
-> experiment was successful, PAL is part of the plasmid pKS2µHyg-PAL-4Cl-CHS
Line 1,984: Line 2,121:
<div class="vector_design">
<div class="vector_design">
-
=== Quick Change mutagenis to insert Ala in front of the Strep - tag II in pYES2_RFC25 MCS ===
+
=== Quick Change mutagenis to insert Ala in front of the Strep - tag II in pTUM104_RFC25 MCS ===
'''Investigator: Ingmar'''
'''Investigator: Ingmar'''
-
Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector; operating purfication possibility via Strep-tag II.  
+
Aim of the experiment: Generation of an RFC 25 compatible version of the pTUM104 Vector; operating purfication possibility via Strep-tag II.  
Operational sequence:
Operational sequence:
Line 1,998: Line 2,135:
2nd picked clone of P44: P46
2nd picked clone of P44: P46
 +
</div>
 +
 +
<div class="thaumatin">
 +
=== Minipreparation of biobricks BBa_J52028, BBa_E2030, BBa_E2020===
 +
 +
'''Investigator: Martin, Alois '''
 +
 +
'''Aim:''' Getting "reporter proteins"
 +
 +
* 10 µl water war added to the well of the distribution kit (=> red)
 +
* BBa_J52028: GFP with PEST191 tag => p51
 +
* BBa_E2030: EYFP, yeast optimized => p52
 +
* BBa_E2020: ECFP, yeast optimized => p53
 +
* Transformation + Minipreparation (Qiagen Plasmid Miniprep Kit)
 +
</div>
</div>
== '''Friday, July 6th''' ==
== '''Friday, July 6th''' ==
<div class="vector_design">
<div class="vector_design">
-
===Quick Change mutagenis to insert Ala in front of the Strep - tag II in pYES2_RFC25 MCS===
+
===Quick Change mutagenis to insert Ala in front of the Strep - tag II in pTUM104_RFC25 MCS===
*Afterwards a control digestion of P45 and P46 was done.
*Afterwards a control digestion of P45 and P46 was done.
Line 2,038: Line 2,190:
[[File:TUM12_gelpicture control digest with PstI 06.07.2012.jpg|500px|Gel picture of control digest with PstI]]
[[File:TUM12_gelpicture control digest with PstI 06.07.2012.jpg|500px|Gel picture of control digest with PstI]]
*All digest products show the expected bonds at 5858 bp. The Miniprep product P45 was chosen to check the insertion of Ala in front of the strep-tag II via DNA sequencing.
*All digest products show the expected bonds at 5858 bp. The Miniprep product P45 was chosen to check the insertion of Ala in front of the strep-tag II via DNA sequencing.
 +
The results of the sequencing are shown below:
 +
 +
[[File:TUM12_Sequencing results of P45.jpg|800px|Sequencing results of P45]]
 +
The sequencing results show that the insertion of Alanin in front of the Strep-tag II was not successful.
</div>
</div>
Line 2,114: Line 2,270:
|1 h
|1 h
|}
|}
 +
</div>
 +
 +
<div class="limonene">
 +
 +
=== Analytic Gel of PCR of Schwab plasmid DNA===
 +
 +
'''Instructor: Andrea'''
 +
 +
[[File:06.07.12.png|400px]]
 +
 +
</div>
 +
 +
<div class="thaumatin">
 +
=== Minipreparation of biobricks BBa_J52028, BBa_E2030, BBa_E2020===
 +
 +
'''Investigator: Martin, Alois '''
 +
 +
'''Aim:''' Getting "reporter proteins"
 +
 +
* 10 µl water war added to the well of the distribution kit (=> red)
 +
* BBa_J52028: GFP with PEST191 tag => p51
 +
* BBa_E2030: EYFP, yeast optimized => p52
 +
* BBa_E2020: ECFP, yeast optimized => p53
 +
* Transformation + Minipreparation (Qiagen Plasmid Miniprep Kit)
 +
 +
</div>
 +
 +
== '''Saturday, July 7th''' ==
 +
<div class="vector_design">
 +
===  Quick Change mutagenesis to insert Ala in front of the Strep-tag II in pTUM104_RFC25 MCS  ===
 +
 +
'''Investigator: Ingmar'''
 +
 +
Aim of the experiment: Generation of an RFC 25 compatible version of the pTUM104 Vector.
 +
*As the sequencing of the first attempt to introduce Ala in front of the Strep-tag II did not show a successfull insertion of Ala, the quickchange mutagense was performed once again with a modified setup. Presumably a formation of primer dimers was responsable for the experiment's results. Therefore the second PCR was operated in two steps as shown below.
 +
'''PCR'''<br>
 +
'''Reaction batch 1'''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|10x Pfu Ultra II buffer
 +
|-
 +
|4 µl
 +
|Plasmid P44 template
 +
|-
 +
|0.5 µl
 +
|1:10 dilution of O54 (10 pmol/µL)
 +
|-
 +
|16.5 µl
 +
|ddH2O
 +
|-
 +
|0.5 µl
 +
|dNTP mix
 +
|-
 +
|0.5 µl
 +
|Pfu Turbo DNA polymerase (2.5 U / µl)
 +
|}
 +
 +
'''Reaction batch 2'''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|10x Pfu Ultra II buffer
 +
|-
 +
|4 µl
 +
|Plasmid P44 template
 +
|-
 +
|0.5 µl
 +
|1:10 dilution of O55 (10 pmol/µL)
 +
|-
 +
|16.5 µl
 +
|ddH2O
 +
|-
 +
|0.5 µl
 +
|dNTP mix
 +
|-
 +
|0.5 µl
 +
|Pfu Turbo DNA polymerase (2.5 U / µl)
 +
|}
 +
 +
'''PCR cycling parameters'''
 +
{|cellspacing="0" border="1"
 +
|'''Segment'''
 +
|'''Cycles'''
 +
|'''Temperature'''
 +
|'''Time'''
 +
|-
 +
|1
 +
|1
 +
|95 °C
 +
| 30 sec
 +
|-
 +
|2
 +
|10
 +
|95°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|55°C
 +
| 1 min
 +
|-
 +
|
 +
|
 +
|67°C
 +
| 6 min
 +
|-
 +
|}
 +
*Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied.
 +
*Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
 +
 +
''' Transformation into ''E.coli'' Xl1-Blue'''
 +
'''Operation Sequence'''
 +
* melting of 100 µl Ca-competent  ''E.coli'' XL1-Blue cells on ice
 +
* addition of 1 µl of the PCR product
 +
* incubation for 30 min on ice
 +
* heat shock for 5 min at 37 °C
 +
* transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
 +
* plate 100 µl on an Amp-LB-plate
 +
* sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
===  Transformation of ''E.coli'' XL1 blue with ADH1 promoter (BBa_J63005), ADH1 terminator(BBa_J63002) and TEF2 promoter from Igem Distribution kit===
 +
 +
'''Investigator: Georg'''
 +
* CaCl2 competent ''E.&nbsp;coli'' XL1-Blue cells were put out from the stock in -80&nbsp;°C freezer and were gently thawed on ice.
 +
 +
* 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
 +
 +
* 30 min incubation on ice
 +
 +
* 5 min. heat shock at 37°C
 +
 +
* Adding of 1ml LB-medium to each tube.
 +
 +
* Incubation for 45min at 37°C in the 180rpm cell-culture shaker.
 +
 +
* 100µl of those cell suspension were plated on ampicillin plates for ADH1-P, and ADH1-T and Kanamycin plates for TEF2-Promoter.
 +
 +
* The rest were centrifuged for 1min at 13000rpm and the supernatant was dicarded.
 +
 +
* The pellet was resuspended in 100µl of LB-medium and this concentrated cell suspension was plated again on new ampicillin- and kanamycin plates.
 +
</div>
 +
</div>
 +
 +
=Week 5=
 +
<!--
 +
<p class="limonene">'''Limonene (4 Experiments):'''</p>
 +
* Ligation of Schwab and BioBricks PCR products in pYES and pSB1C3
 +
 +
<p class="coumaryl">'''Xanthohumol (10 Experiments):'''</p>
 +
* Repetition of PCR with PAL
 +
* Transformation with PCR products of OMT, 4Cl and CHS
 +
 +
<p class="thaumatin">'''Thaumatin (1 Experiment):'''</p>
 +
* Preparation of YPD medium
 +
 +
<p class="constitutive_promoter">'''Constitutive Promoter (10 Experiments):'''</p>
 +
* Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder
 +
*
 +
 +
<p class="light_switchable_promoter">'''Light Switchable Promoter (14 Experiments):'''</p>
 +
*
 +
 +
<html><a class="WDetails" href="#Week_5" id="Week_5">Show Details ...</a></html>
 +
-->
 +
<div class="week" id="WWeek_5">
 +
== '''Monday, July 9th''' ==
 +
<div class="constitutive_promoter">
 +
=== Picking of colonies of TEF2-P, ADH1-P, ADH1-T (iGEM)===
 +
'''Investigator:''' Georg
 +
 +
* To 5 µl LB-Medium,5 µl of 1000x stock of ampicillin (ADH-P, ADH1-T) and kanamycin (TEF2-P) were added
 +
* 4 colonies from the plate with TEF2-P and 5 colonies of ADH1-P and ADH1-T were picked
 +
* Each colony was transferred into 5 ml LB-Medium with 1x ampicillin or kanamycin
 +
* Incubation over night at 37°C in the 180rpm cell-culture shaker.
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
===Repetition of picking of colonies of ADH1-P, ADH1-T, TEF2-P from iGEM distribution kit===
 +
 +
'''Investigator''': Georg
 +
 +
* Analytical Gel was loaded with 9 µl digestion and 1 µl 10x buffer
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
=== Repetition of picking of colonies of TEF2-P, ADH1-P, ADH1-T (iGEM)===
 +
'''Investigator:''' Georg
 +
*Only ADH1 promoter showed cell proliferation. The rest showed no proliferation.The reason was a 10x too high amount of antibiotics.
 +
 
 +
*Colonies from  transformation from ADH1-promoter and terminator  were picked again and incubated in LB medium with 
 +
ampicillin at a dilution of 1:1000 and for TEF2 –Promoter with Kanamycin at a dilution of 1:1000
 +
*From the grown colonies from the transformation with ADH1-Promoter then were the plasmids extracted, using the
 +
Quiaprep Spin Miniprep kit from Quiagen
 +
*The extracted ADH1-P DNA was then analytically digested  with XbaI and PstI from Fermentas
 +
 +
* Reaction batch for digestion:
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1 µl
 +
|PSTI (Fermentas)
 +
|-
 +
|1 µl 
 +
|XbaI (Fermentas)
 +
|-
 +
|8 µl
 +
|Tango buffer
 +
|-
 +
|60µl
 +
|dd H20
 +
|-
 +
|=70µl
 +
|'''TOTAL'''
 +
|}
 +
* To 17,5 µl mastermix was 2,5 µl of plasmid DNA added
 +
 +
* to 2,5 ml of DNA 17,5 µl reaction batch were added
 +
* Digestion took place at 37°C for 1 h
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Phycocyanobilin (PCB) extraction from dried ''Spirulina platensis'' powder (part 1/4) ===
 +
'''Investigator: Jeff, Alois, Martin'''
 +
 +
'''Aim of the experiment:''' Phycocyanobilin (PCB) is a cofactor neeeded for the funtion of phytochrome B. Phycocyanobilin is covalently bound to Cys457 of phytochrome B. ''Saccharomyces cerevisiae'' does not contain endogenous PCB. For proof of concept PCB should be added to the medium. In the following experiment, PCB is extracted from dried ''Spirulina platensis'' powder.
 +
 +
'''Operational sequence:'''
 +
* 50&nbsp;g of ''Spirulina platensis'' powder was (from concept-vitalprodukte.de) resuspended in 1.5&nbsp;l of H2O (30&nbsp;mg/l) in a beaker covered with aluminium foil.
 +
 +
* Stirring for 10&nbsp;min at RT.
 +
 +
* Green ''Spirulina'' suspension was divided in 6 centrifuge bottles, covered in aluminium foil.
 +
 +
* Centrifation at 10500&nbsp;rpm at 4&nbsp;°C (SLA-3000 rotor, Thermo Scientific) for 1&nbsp;h.
 +
 +
* Supernatant was discarded
 +
 +
* 25&nbsp;ml of MeOH added to each bottle and was heavily shaked to resuspend the pellet for the next cleaning step with MeOH.
 +
 +
* Each bottle with the resuspended pellet were filled with MeOH to a final volume of 250&nbsp;ml and shaked again to fully resuspend the pellet.
 +
 +
* Centrifugation at 10500&nbsp;rpm at 4&nbsp;°C (SLA-3000 rotor, Thermo Scientific) for 10&nbsp;min.
 +
 +
* Supernatant was discarded.
 +
 +
* The last 4 steps were repeated until the supernatant of the washed pellet was colorless or cyanblue but not green anymore (Regulary, it takes 3 or 4 times). Pellet should be cyanblue now.
 +
 +
* The pellet of the 6 centrifuge tubes was collected in a sole centrifugation tube, covered in aluminium foil tube, by scratching it out with a small spoon.
 +
 +
* The remaining rest of the pellet which cannot be scratched out were resuspended in a small amount of MeOH and were transformed from tube to tube with a interim shaking step.
 +
 +
* This suspension was transferred into the tube with the scratched-out pellet.
 +
 +
* Centrifugation at 10500&nbsp;rpm at 4&nbsp;°C (SLA-3000 rotor, Thermo Scientific) for 10&nbsp;min.
 +
 +
* Supernatant was discarded.
 +
 +
* Finally washed pellet was stored, wrapped in foil overnight for the methanolysis next day.
 +
 +
[[File:TUM12_LSPS_WP_000736.jpg|500px|Green dried ''Spirulina platensis'' powder]]
 +
[[File:TUM12_LSPS_WP_000716.jpg|500px|Cyanblue Pellet after 4x of MeOH washing step]]
 +
[[File:TUM12_LSPS_WP_000720.jpg|500px|Cyanblue supernatant of the last washing step with MeOH. It indicates that all the green chromophores are already washed out, which means that only the protein-bound phycocyanobilin is still arrested in he pellet]]
 +
[[File:TUM12_LSPS_WP_000717.jpg|500px|Resuspended Pellet in MeOH to pool all the pellet]]
 +
[[File:TUM12_LSPS_WP_000734.jpg|500px|Analytical sample of the dried pellet before the methanolysis for later analysis]]
 +
</div>
 +
 +
== '''Tuesday, July 10th''' ==
 +
<div class="constitutive_promoter">
 +
===Analytical digestion of ADH1-P and gelelectrophoresis===
 +
''' Investigator:''' Georg
 +
 +
* Reaction batch for digestion:
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1 µl
 +
|PstI-HF (NEB)
 +
|-
 +
|1 µl 
 +
|XbaI (NEB)
 +
|-
 +
|8 µl
 +
|NEB-4 buffer
 +
|-
 +
|0,8 µl
 +
|100 x BSA (NEB)
 +
|-
 +
|59,2 µl
 +
|dd H20
 +
|-
 +
|=70µl
 +
|'''TOTAL'''
 +
|}
 +
 +
[[File:10.07.2012 ADH1 Prom (fertig).PNG]]
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
 +
===Extraction of ADH1-P, TEF2-T with plasmid===
 +
 +
'''Investigator:''' Georg
 +
 +
* Plasmid-DNA was extracted according to Quiaprep Plasmid extraction kit
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
===Inoculation of TEF1-P, PGK-P, Cyc-T===
 +
 +
'''Investigator:''' Georg
 +
 +
* Transfomed E. coli cells from iGEM were inoculated onto Amp-LB-Plates in case of PGK1-P and Cyc1-T
 +
* E. coli with TEF1-T were inoculated onto psb1c3
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Phycocyanobilin (PCB) extraction from dried ''Spirulina platensis'' powder (part 2/4) ===
 +
'''Investigator: Jeff, Alois, Martin'''
 +
 +
'''Aim of the experiment:''' Phycocyanobilin (PCB) is a cofactor neeeded for the funtion of phytochrome B. Phycocyanobilin is covalently bound to Cys457 of phytochrome B. ''Saccharomyces cerevisiae'' does not contain endogenous PCB. For proof of concept PCB should be added to the medium. In the following experiment, PCB is extracted from dried ''Spirulina platensis'' powder.
 +
 +
'''Operational sequence:'''
 +
 +
* Washed pellet from the day before was resuspended in 500&nbsp;ml MeOH.
 +
 +
* Heat suspension in a 500&nbsp;ml flask in a water bath at 70 – 75&nbsp;ºC with a condensing coil cooled with water for 5 – 8 hrs.
 +
 +
* After this, the suspension was transferred into a new centrifuge tube and centrifuged at 10500&nbsp;rpm at 4&nbsp;°C (SLA-3000 rotor, Thermo Scientific) for 20&nbsp;min.
 +
 +
* The supernatant was decanted trough a filter paper into new centrifugation tube and stored, wrapped in a aluminium foil, at -20&nbsp;°C.
 +
 +
* The pellets also was stored, wrapped in a aluminium foil, at -20&nbsp;°C.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Plating of received ''E.&nbsp;coli'' containing biobricks ===
 +
'''Investigator: Jeff'''
 +
 +
'''Aim of the experiment:''' The received biobricks were already transformed in ''E.&nbsp;coli'' and were in an agar stabs. These ''E.&nbsp;coli'' cells were transferred with an inoculation loop on antibiotic selection plates and were incubated over night.
 +
 +
'''Operational sequence:'''
 +
* Bacterias containing plasmids with biobricks were transferred with a sterile inoculation loop on antibiotic plates and were incubated at 37&nbsp;°C overnight.
 +
 +
* The biobricks were: '''BBa_K207001''' in pSB1A2, '''BBa_K243006''' in BBa_K157000, '''BBa_K300001''' in K300007 (part for other subproject), '''BBa_K268000''' in pSB6A0 (part for other subproject), '''BBa_K365005''' RFC25 in pSB1C3, '''BBa_K365000''' in pSB1C3, '''BBa_K207000''' in pSB3K3, '''BBa_K165031''' in pSB1AK3
 +
</div>
 +
 +
== '''Wednesday, July 11th''' ==
 +
<div class="constitutive_promoter">
 +
===Analytical digestion and gelelectrophoresis of ADH1-Terminator and TEF2-P===
 +
 +
'''Investigator:''' Georg
 +
 +
* Transformants of ADH1-T showed no Plasmid
 +
* TEF2 Promoter-colonies were all positive
 +
 +
[[File:20120711 tef2promotor adh1terminator.PNG]]
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
=== Picking of inoculated CYC1-T, TEF1-P, PGK1-P for overnight cultures===
 +
 +
'''Investigator:''' Georg
 +
 +
* To 5 µl LB-Medium,5 µl of 1000x Chloramphenicol were added
 +
* 4 colonies from the plate with TEF1-P, 4 colonies of Cyc-T were picked and 4 colonies of PGK1-P were picked
 +
* Each colony was transferred into 5 ml LB-Medium with 1x Chloramphenicol (TEF1-P)or Amp (PGK1-P, Cyc1-T)
 +
* Incubation over night at 37°C in the 180rpm cell-culture shaker.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Phycocyanobilin (PCB) extraction from dried ''Spirulina platensis'' powder (part 3/4) ===
 +
'''Investigator: Jeff, Alois, Martin'''
 +
 +
'''Aim of the experiment:''' Phycocyanobilin (PCB) is a cofactor neeeded for the funtion of phytochrome B. Phycocyanobilin is covalently bound to Cys457 of phytochrome B. ''Saccharomyces cerevisiae'' does not contain endogenous PCB. For proof of concept PCB should be added to the medium. In the following experiment, PCB is extracted from dried ''Spirulina platensis'' powder.
 +
 +
'''Operational sequence:'''
 +
 +
* The pellet after the first methanolysis of the day before was undergone a second methanolysis step to ensure high efficiacy of PCB extraction. Operational sequence was performed like the first methanolysis including the centrifugation and filtering step. The pellet after the second methanolysis should be more colorless and the filtered supernatant was freezed, like the one from the first methanolysis, at -20&nbsp;°C, wrapped in aluminium foil
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Transformation of ''E.&nbsp;coli'' XL1-Blue with pGADT7 and pGBKT7 plasmid for Yeast-two-hybrid (Y2H) ===
 +
'''Investigator: Jeff'''
 +
 +
'''Aim of the experiment:''' pGADT7 and pGBKT7 are plasmids containing the transcriptional activation domain or the DNA binding domain of the transcription activator Gal4. pGADT7 contains the transcriptional activation domain of gal4; we want to clone the the first 100 residues of Pif3 (BBa_K365000) into this plasmid. As a result we have a fusion contruct of Gal4 and Pif3, which is nescassary for the light-switchable promoter system. pGBKT7 is for backup, if the ordered biobricks are not working.
 +
 +
'''Operational sequence:'''
 +
 +
* ''E.&nbsp;coli'' XL1-Blue are transformed with pGADT7 and pGBKT7 seperately after standard protocol of our lab.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Introducing new ''Saccharomyces cerevisiae'' strain (Y190 strain) from Schwab lab for Y2H ===
 +
'''Investigator: Jeff'''
 +
 +
'''Aim of the experiment:''' The Y190 ''Saccharomyces cerevisiae'' is a special strain for Yeast-two-hybrid. This strain carries a Gal4 and Gal80 deletion to higher the signal/noise-ratio of protein-protein interactions. Reporter for protein-protein interactions are HIS3, lacZ and MEL1 and are encoded in the genomic DNA. Transformation markers are trp1, leu2 and cyhR2 and are encoded on the transformation plasmids.
 +
 +
'''Operational sequence:'''
 +
* The freshly plated Y190 cells are transferred with a inoculation loop from the original plate on a new YPD agar plate.
 +
 +
* After 2 days the the plate was put in 4&nbsp;°C.
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
=== Repetition of PCR of PAL===
 +
 +
'''Investigator: Katrin, Daniela'''
 +
 +
'''Reaction batch'''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5 µl
 +
|10x Pfu Ultra II buffer
 +
|-
 +
|4 µl
 +
|dNTP's (each 2.5 mM)
 +
|-
 +
|0.5 µl
 +
|Pfu Ultra II (2.5 U/µL)
 +
|-
 +
|5 µl
 +
|1:10 dilution of used forward primers (10µM)
 +
|-
 +
|5 µl
 +
|1:10 dilution of used reversed primers (10µM)
 +
|-
 +
|1 µl
 +
|DNA (pKS2µHyg-PAL-4CL-CHS 50 ng/µL)
 +
|-
 +
|29.5 µL
 +
|bidest. sterile Water
 +
|}
 +
 +
'''PCR cycling parameters'''
 +
{|cellspacing="0" border="1"
 +
|'''Segment'''
 +
|'''Cycles'''
 +
|'''Temperature'''
 +
|'''Time'''
 +
|-
 +
|1
 +
|1
 +
|95 °C
 +
| 2 min (and adding Pfu Ultra after 2 min)
 +
|-
 +
|2
 +
|30
 +
|95°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|52°C
 +
| 1 min
 +
|-
 +
|
 +
|
 +
|72°C
 +
| 2.5 min
 +
|-
 +
|3
 +
|
 +
|72°C
 +
| 5 min
 +
|-
 +
|}
 +
 +
'''PCR purification'''
 +
*Purification was done using QIAquick PCR Purification Kit (250)
 +
 +
'''Analytical gelelektrophoresis of PCR-Products of PAL'''
 +
 +
-> PCR was not successful, no band at 2,1 kb
 +
(picture follows)
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
=== Preparative Digest of pYES2_iGEM===
 +
 +
'''Investigator: Katrin, Daniela'''
 +
 +
'''Digestion of P50 with Xbal and NgOMIV'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|10 µl
 +
|P50 (concentration: 264.5 ng/µl)
 +
|-
 +
|2.5 µl
 +
|NEB4
 +
|-
 +
|2.5 µl
 +
|10x BSA
 +
|-
 +
|1 µl
 +
|XbaI (20 U/µl)
 +
|-
 +
|2 µl
 +
|NgOMIV (10U/µl)
 +
|-
 +
|7 µl
 +
|ddH2O
 +
|}
 +
 +
Incubation: 37 °C, 3 h
 +
 +
'''Digestion of P50 with Xbal and PstI'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|10 µl
 +
|P50 (concentration: 264.5 ng/µl)
 +
|-
 +
|5 µl
 +
|Tango buffer
 +
|-
 +
|2 µl
 +
|XbaI
 +
|-
 +
|3 µl
 +
|PstI
 +
|-
 +
|7.5 µl
 +
|ddH2O
 +
|}
 +
 +
Incubation: 37 °C, 3 h
 +
 +
'''DNA preparative gel electrophoresis'''
 +
*gel: 1% with LMP-agarose
 +
*band 1: P50 digested with XbaI and PstI
 +
*band 2: P50 digested with XbaI and NgOMIV
 +
*70 V, 90 min
 +
 +
'''Gelextration'''
 +
*cut the bands
 +
*QIAquick Gel Extractrion Kit was used
 +
** step 6 was left out
 +
** step 9: 30µl buffer, 4 min incubation
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
=== Ligation of digested P50 with digested PCR-products of PCR 15-20 (4CL, CHS and OMT)===
 +
 +
'''Investigator: Katrin, Daniela'''
 +
 +
Concentration (Nano Drop: <br />
 +
4CL+ (PCR 15) = 40.3 ng/µl<br />
 +
4CL- (PCR 16) = 37.3 ng/µl<br />
 +
CHS+ (PCR 17) = 46.1 ng/µl<br />
 +
CHS- (PCR 18) = 51.2 ng/µl<br />
 +
OMT+ (PCR 19) = 22.3 ng/µl<br />
 +
OMT- (PCR 20) = 16.7 ng/µl<br />
 +
<br />
 +
P50 digested with XbaI and PstI = 23.9 ng/µl (the digested Plasmid has the number P71)<br />
 +
P50 digested with XbaI and NgOMIV = 28.4 ng/µl (the digested Plasmid has the number P72)<br />
 +
<br />
 +
*required volumes were calculated using Lab Tools
 +
4CL+ (PCR 15) with P50 digested with XbaI and PstI
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5.27 µl
 +
|P50 digested
 +
|-
 +
|2.73 µl
 +
|4CL+ (PCR 15)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
 +
4CL- (PCR 16) with P50 digested with XbaI and PstI
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5.13 µl
 +
|P50 digested
 +
|-
 +
|2.87 µl
 +
|4CL- (PCR 16)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
CHS+ (PCR 17) with P50 digested with XbaI and NgOMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5.84 µl
 +
|P50 digested
 +
|-
 +
|2.16 µl
 +
|CHS+ (PCR 17)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
CHS- (PCR 18) with P50 digested with XbaI and NgOMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|6 µl
 +
|P50 digested
 +
|-
 +
|2 µl
 +
|CHS- (PCR 18)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
OMT+ (PCR 19) with P50 digested with XbaI and NgOMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.45 µl
 +
|P50 digested
 +
|-
 +
|3.55 µl
 +
|OMT+ (PCR 19)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
OMT- (PCR 20) with P50 digested with XbaI and NgOMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|3.87 µl
 +
|P50 digested
 +
|-
 +
|4,13 µl
 +
|OMT- (PCR 20)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
Negative control
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5 µl
 +
|P50 digested with XbaI and PstI or NgOMIV
 +
|-
 +
|3 µl
 +
|ddH2O
 +
|-
 +
|1 µl
 +
|T4 DNA-ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
 +
*water bath 16 °C
 +
 +
</div>
 +
 +
== '''Thursday, July 12th''' ==
 +
<div class="constitutive_promoter">
 +
=== Gelextraction and analytical digestion of CYC1-terminator, TEF1-promoter, and PGK1-Promoter from pSB1C3 and pSB1A2===
 +
 +
'''Investigator: Georg'''
 +
 +
*Genes from overnight cultures were extracted, using the Quiaprep gene extraction Kit. Extracted Plasmids then were analytically digested with XbaI and PstI (Fermentas).
 +
 +
'''Analytical digestion XbaI, PstI
 +
{|Cellspacing="O", border="1"
 +
|'''Chemical'''
 +
|'''Volume'''
 +
|-
 +
|XbaI
 +
|2,5 µl
 +
|-
 +
|PstI
 +
|2,5 µl
 +
|-
 +
|10xBuffer Tango
 +
|2 µl
 +
|-
 +
|Plasmid-DNA
 +
|2,5 µl
 +
|-
 +
|ddH2O
 +
|165,5 µl
 +
|-
 +
|=175µl
 +
|'''TOTAL'''
 +
|}
 +
 +
*Analytical gel was run (1% Agarose).
 +
 +
[[File:20120712-PGK1-P,-Cyc-T,-TEf.png]]
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Phycocyanobilin (PCB) extraction from dried ''Spirulina platensis'' powder (part 4/4) ===
 +
'''Investigator: Martin, Jeff, Alois'''
 +
 +
'''Aim of the experiment:''' Phycocyanobilin (PCB) is a cofactor neeeded for the funtion of phytochrome B. Phycocyanobilin is covalently bound to Cys457 of phytochrome B. ''Saccharomyces cerevisiae'' does not contain endogenous PCB. For proof of concept PCB should be added to the medium. In the following experiment, PCB is extracted from dried ''Spirulina platensis'' powder.
 +
 +
'''Operational sequence:'''
 +
 +
* The supernatants (~1l in total) of the two previous experiments were pooled and put into a rotary evaporater in order to acquire a concentrate of approximately 100ml.
 +
* The settings of the rotary evaporater: ~160 millibars, waterbath temperature of 25-30°C
 +
* Furthermore, there were measures to be taken to protect the solution from direct sunlight: blinds down, aluminum foil wrapped around the waterbath
 +
 +
* Afterwards we transfered the Methanol solution into a separating funnel and added another 100ml of aqua dest..
 +
* By means of adding chloroform we created two phases. The lower (chloroform) phase with the solved Phycocyanobilin was separated and put into another rotary evaporater flask. This step was repeated three times in order to get all the Phycocanobilin into the next step
 +
 +
* Then the chloroform was completely removed in the rotary evaporater (same settings as before), the pure (?) Phycocyanobilin in the flask was solved in 60 ml DMSO, transfered into a new flask and frozen at -20&nbsp;°C.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Picking of transformated (pGADT7 and pGBKT7) ''E.coli cells'' on antibiotic selection plates  ===
 +
'''Investigator: Jeff'''
 +
 +
'''Aim of the experiment:''' pGADT7 and pGBKT7 are plasmids containing the transcriptional activation domain or the DNA binding domain of the transcription activator Gal4. pGADT7 contains the transcriptional activation domain of gal4; we want to clone the the first 100 residues of Pif3 (BBa_K365000) into this plasmid. As a result we have a fusion contruct of Gal4 and Pif3, which is nescassary for the light-switchable promoter system. pGBKT7 is for backup, if the ordered biobricks are not working.
 +
 +
'''Operational sequence:'''
 +
* A ''E.&nbsp;coli'' colony was picked for each plasmid and was transferred in a tube containing 4&nbsp;ml of LB-medium containing antibiotics (Amp for pGADT7 and Kan for pGBKT7).
 +
 +
* Overnight culture at 37&nbsp;°C in a cell-culture shaker.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Miniprep of transformated ''E.coli'' from overnight culture (8 plasmids containing biobricks)  ===
 +
'''Investigator: Andrea'''
 +
 +
'''Aim of the experiment: Miniprep of transformated E.&nbsp;coli from overnight culture to get the plasmids with biobricks'''
 +
 +
'''NanoDrop Measure'''
 +
{|cellspacing="0" border="1"
 +
|'''Plasmid'''
 +
|'''Concentration'''
 +
|-
 +
|P73
 +
|41.6&nbsp;ng/µl
 +
|-
 +
|P74
 +
|85.2&nbsp;ng/µl
 +
|-
 +
|P75
 +
|72.4 ng/µl
 +
|-
 +
|P76
 +
|254.3 ng/µl
 +
|-
 +
|P77
 +
|74.4 ng/µl
 +
|-
 +
|P78
 +
|50.0 ng/µl
 +
|-
 +
|P79
 +
|46.2 ng/µl
 +
|-
 +
|P80
 +
|116.7 ng/µl
 +
|-
 +
|P81
 +
|77.1 ng/µl
 +
|-
 +
|P82
 +
|65.5 ng/µl
 +
|}
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Analytical digestion and gelelectrophoresis of biobricks (8 plasmids containing biobricks) ===
 +
'''Investigator: Jeff'''
 +
 +
'''Aim of the experiment''': Checking whether the biobricks are part of the plasmid-backbone.
 +
 +
'''Operational sequence:'''
 +
 +
* Reaction batch for each plasmid:
 +
{|cellspacing="0" border="1"
 +
|'''Reagent'''
 +
|'''Volume in µl'''
 +
|-
 +
|Tango Buffer 10x
 +
|4&nbsp;µl
 +
|-
 +
|XbaI (Fermentas)
 +
|0.25&nbsp;µl
 +
|-
 +
|PstI (Fermentas)
 +
|0.5&nbsp;µl
 +
|-
 +
|Plasmid DNA
 +
|2.5&nbsp;µl
 +
|-
 +
|ddH2O
 +
|12.75&nbsp;µl
 +
|-
 +
|'''TOTAL'''
 +
|'''20&nbsp;µl'''
 +
|}
 +
 +
* Incubation at 37&nbsp;°C for 1&nbsp;h 45&nbsp;min.
 +
 +
* Analytical gelelectrophoresis at 90&nbsp;V for 1&nbsp;h.
 +
 +
* Order of gel-pockets:
 +
{|cellspacing="0" border="1"
 +
|100&nbsp;bp ladder
 +
|P82
 +
|P81
 +
|P80
 +
|P79
 +
|P76
 +
|P75
 +
|P74
 +
|P73
 +
|1&nbsp;kbp ladder
 +
|-
 +
|
 +
|OK
 +
|BAD
 +
|OK
 +
|OK
 +
|OK
 +
|OK
 +
|OK
 +
|BAD
 +
|
 +
|}
 +
 +
* P73 and P81, both parts from Havard university are bad. To exclude errors, one should pick another colony for each plasmid and do again the analytical digestion and gelelectrophoresis after the miniprep.
 +
 +
[[File:TUM12_20120712_2.jpg|500px|Analytical gelelectrophoresis with XbaI and PstI]]
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
=== Transformation of Ligationproducts of pTUM104 + OMT, 4Cl and CHS in ''E.coli''===
 +
 +
'''Investigator: Mary'''
 +
 +
* adding 5µl ligation product in 100µl competent XL blue ''E.coli'' cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (Withouth antibiotica) and incubate at 37°C, 30 min, 180 rpm
 +
* plate on agar with Ampicillin over night
 +
</div>
 +
 +
<div class="coumaryl">
 +
=== Repetition of PCR of PAL===
 +
 +
'''Investigator: Daniela'''
 +
 +
Aim of the experiment: Repetition of PCR of PAL (so far not successful) with the use of different polymerase and try of 3 temperatures
 +
 +
only with PAL+, 3 different temperatures (see cycling parameters) and one batch at 52.5 °C with DMSO
 +
 +
'''Reaction batch'''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|10 µl
 +
|5x Herculase II reaction buffer
 +
|-
 +
|0,5 µl
 +
|dNTP Mix (each dNTP 2.5 mM)
 +
|-
 +
|1 µl
 +
|Herculase II fusion DNA Polymerase
 +
|-
 +
|1,25 µl
 +
|1:10 dilution of used forward primers (O22)
 +
|-
 +
|1,25 µl
 +
|1:10 dilution of used reversed primers (O59)
 +
|-
 +
|1 µl
 +
|1:10 dilution of DNA (P19=pKS2µHyg-PAL-4CL-CHS 50 ng/µL) -> 5 ng/µL
 +
|-
 +
|35 µL
 +
|ddH2O
 +
|}
 +
 +
'''Reaction batch with DMSO'''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|10 µl
 +
|5x Herculase II reaction buffer
 +
|-
 +
|0,5 µl
 +
|dNTP Mix (each dNTP 2.5 mM)
 +
|-
 +
|1 µl
 +
|Herculase II fusion DNA Polymerase
 +
|-
 +
|1,25 µl
 +
|1:10 dilution of used forward primers (O22)
 +
|-
 +
|1,25 µl
 +
|1:10 dilution of used reversed primers (O59)
 +
|-
 +
|1 µl
 +
|1:10 dilution of DNA (pKS2µHyg-PAL-4CL-CHS 50 ng/µL) -> 5 ng/µL
 +
|-
 +
|1,5 µl
 +
|DMSO (3% des Ansatzes)
 +
|-
 +
|33.5 µL
 +
|ddH2O
 +
|}
 +
 +
'''PCR cycling parameters'''
 +
{|cellspacing="0" border="1"
 +
|'''Segment'''
 +
|'''Cycles'''
 +
|'''Temperature'''
 +
|'''Time'''
 +
|-
 +
|1
 +
|1
 +
|95 °C
 +
| 2 min
 +
|-
 +
|2
 +
|30
 +
|95°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|52.5°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|72°C
 +
| 4 min
 +
|-
 +
|3
 +
|
 +
|72°C
 +
| 3 min
 +
|-
 +
|}
 +
 +
'''PCR cycling parameters'''
 +
{|cellspacing="0" border="1"
 +
|'''Segment'''
 +
|'''Cycles'''
 +
|'''Temperature'''
 +
|'''Time'''
 +
|-
 +
|1
 +
|1
 +
|95 °C
 +
| 2 min
 +
|-
 +
|2
 +
|30
 +
|95°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|45°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|72°C
 +
| 4 min
 +
|-
 +
|3
 +
|
 +
|72°C
 +
| 3 min
 +
|-
 +
|}
 +
 +
'''PCR cycling parameters'''
 +
{|cellspacing="0" border="1"
 +
|'''Segment'''
 +
|'''Cycles'''
 +
|'''Temperature'''
 +
|'''Time'''
 +
|-
 +
|1
 +
|1
 +
|95 °C
 +
| 2 min
 +
|-
 +
|2
 +
|30
 +
|95°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|56.2°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|72°C
 +
| 4 min
 +
|-
 +
|3
 +
|
 +
|72°C
 +
| 3 min
 +
|-
 +
|}
 +
Analytical gel electrophoresis
 +
*no bands at all (picture follows)
 +
*possibly due to low concentration of P19 (5 ng/µl)
 +
</div>
 +
<div class="coumaryl">
 +
 +
=== Ligation of P93 (pSB1C3 digested with XbaI and AgeI) with digested PCR-products of PCR 17-20 (CHS and OMT)===
 +
 +
'''Investigator: Daniela'''
 +
 +
Concentration (Nano Drop: <br />
 +
CHS+ (PCR 17) = 46.1 ng/µl<br />
 +
CHS- (PCR 18) = 51.2 ng/µl<br />
 +
OMT+ (PCR 19) = 22.3 ng/µl<br />
 +
OMT- (PCR 20) = 16.7 ng/µl<br />
 +
<br />
 +
P93 (digested with XbaI and AgeI) = 4.8 ng/µl<br />
 +
 +
<br />
 +
CHS+ (PCR17) with P93 digested with XbaI and AgeI
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|7.04 µl
 +
|P93
 +
|-
 +
|0.96 µl
 +
|CHS+ (PCR 17)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
CHS- (PCR 18) with P93 digested with XbaI and AgeI
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|7.13 µl
 +
|P93
 +
|-
 +
|0.87 µl
 +
|CHS- (PCR18)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
OMT+ (PCR19) with P93 digested with XbaI and AgeI
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|6.19 µl
 +
|P93 
 +
|-
 +
|1.81 µl
 +
|OMT+ (PCR19)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
OMT- (PCR20) with P50 digested with XbaI and AgeI
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5.75 µl
 +
|P93 
 +
|-
 +
|2.25 µl
 +
|OMT- (PCR20)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
Negative control
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5 µl
 +
|P93 
 +
|-
 +
|3 µl
 +
|ddH2O
 +
|-
 +
|1 µl
 +
|T4 DNA-ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
 +
*water bath 16 °C
 +
*products were named P94-P97
 +
 +
</div>
 +
<div class="limonene">
 +
===Preparative digest of PCR1-PCR7 and P50===
 +
'''Investigator: Andrea'''
 +
 +
'''Digestion of P50 with XbaI and NgoMIV'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|20 µl
 +
|P50
 +
|-
 +
|4 µl
 +
|NEB Buffer
 +
|-
 +
|0.4 µl
 +
|BSA
 +
|-
 +
|1 µl
 +
|XbaI (10 U/µl)
 +
|-
 +
|2 µl
 +
|NgoMIV (10 U/µl)
 +
|-
 +
|27.4 µl
 +
|ddH2O
 +
|}
 +
 +
'''Digestion of PCR1-PCR7 with XbaI and AgeI'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|25 µl
 +
|PCR-Product
 +
|-
 +
|5 µl
 +
|NEB Buffer
 +
|-
 +
|0.5 µl
 +
|BSA
 +
|-
 +
|1 µl
 +
|XbaI (10 U/µl)
 +
|-
 +
|1 µl
 +
|AgeI (10 U/µl)
 +
|-
 +
|32.5 µl
 +
|ddH2O
 +
|}
 +
 +
Incubation: 37 °C, 3 h
 +
 +
</div>
 +
 +
<div class="limonene">
 +
=== Preparative gel electrophoresis of digested plasmids PCR1-PCR7 and PCR9 ===
 +
 +
'''Investigator: Andrea'''
 +
 +
'''Aim of the experiment:''' Analytical gel electrophoresis of products from restriction digest of plasmids PCR1 - PCR7 and PCR9
 +
 +
Limonensynthase: 1600 bp
 +
 +
[[File:12.07.12 prep digest1.png|400px]]
 +
 +
[[File:12.07.12_prep_digest2.png|400px]]
 +
 +
[[File:12.07.12_prep_digest3.png|400px]]
 +
 +
</div>
 +
 +
<div class="limonene">
 +
 +
=== Ligation of digested PCR1-PCR7 (digested with XbaI and AgeI) with pSB1C3 (digested with XbaI and AgeI) and with pYESnew (digested with XbaI and NgoMIV)===
 +
 +
'''Investigator: Andrea'''
 +
 +
Concentration (Nano Drop: <br />
 +
LIMS Citrus (PCR 1) = 13.4 ng/µl<br />
 +
LIMS Citrus (PCR 2) = 10.3 ng/µl<br />
 +
LIMS Lavendula (PCR 3) = 2.2 ng/µl<br />
 +
LIMS Lavendula (PCR 3) = 9.9 ng/µl<br />
 +
LIMS Lavendula (PCR 3) = 9.6 ng/µl<br />
 +
LIMS Lavendula (PCR 3) = 9.8 ng/µl<br />
 +
LIMS Lavendula (PCR 3) = 12.3 ng/µl<br />
 +
<br />
 +
P50 (digested with XbaI and NgoMIV) = 10.3 ng/µl<br />
 +
<br />
 +
P93 (digested with XbaI and AgeI) = 4 ng/µl<br />
 +
 +
<br />
 +
PCR1 with P50 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.89µl
 +
|P50
 +
|-
 +
|3.11 µl
 +
|PCR 1
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR2 with P50 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.38 µl
 +
|P50
 +
|-
 +
|3.62 µl
 +
|PCR2
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR3 with P50 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.64 µl
 +
|P50 
 +
|-
 +
|6.36 µl
 +
|PCR3
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR4 with P50 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.3 µl
 +
|P50 
 +
|-
 +
|3.7 µl
 +
|PCR4
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR5 with P50 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.26 µl
 +
|P50 
 +
|-
 +
|3.74 µl
 +
|PCR5
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR6 with P50 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.26 µl
 +
|P50 
 +
|-
 +
|3.74 µl
 +
|PCR6
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR7 with P50 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|7.83 µl
 +
|P50 
 +
|-
 +
|3.27 µl
 +
|PCR7
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
Negative control
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5 µl
 +
|P50 
 +
|-
 +
|3 µl
 +
|ddH2O
 +
|-
 +
|1 µl
 +
|T4 DNA-ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
<br />
 +
PCR1 with P93 digested with XbaI and AgeI
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.89 µl
 +
|P93 
 +
|-
 +
|3.11 µl
 +
|PCR1
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR2 with P93 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.38 µl
 +
|P93 
 +
|-
 +
|3.62 µl
 +
|PCR2
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR3 with P93 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.52 µl
 +
|P93 
 +
|-
 +
|6.48 µl
 +
|PCR3
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR4 with P93 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.3 µl
 +
|P93 
 +
|-
 +
|3.7 µl
 +
|PCR4
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR5 with P93 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.26 µl
 +
|P93 
 +
|-
 +
|3.74 µl
 +
|PCR5
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR6 with P93 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.26 µl
 +
|P93 
 +
|-
 +
|3.74 µl
 +
|PCR6
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
PCR7 with P93 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.72 µl
 +
|P93 
 +
|-
 +
|3.28 µl
 +
|PCR7
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
 +
*water bath 16 °C
 +
*products were named P100-P115
 +
 +
</div>
 +
 +
== '''Friday, July 13th''' ==
 +
<div class="constitutive_promoter">
 +
=== Transformation of ADH1-T ===
 +
 +
'''Procedure:'''
 +
 +
'''Investigator''': Georg
 +
 +
* CaCl2 competent ''E.&nbsp;coli'' XL1-Blue cells were put out from the stock in -80&nbsp;°C freezer and were gently thawed on ice.
 +
 +
* 2 µl of ADH-T plasmid was added to 100 µl of competent cells and gently mixed.
 +
 +
* 30 min incubation on ice
 +
 +
* 5 min. heat shock at 37°C
 +
 +
* Adding of 1ml LB-medium to each tube.
 +
 +
* Incubation for 45min at 37°C in the 180rpm cell-culture shaker.
 +
 +
* 100µl of those cell suspension were plated on ampicillin plates.
 +
 +
* The rest were centrifuged for 1min at 13000rpm and the supernatant was dicarded.
 +
 +
* The pellet was resuspended in 100µl of LB-medium and this concentrated cell suspension was plated again on new ampicillin plates.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== PCR of P73, P80 and P81 to add RFC25 pre- and suffix ===
 +
 +
'''Investigator:''' Jeff, Saskia
 +
 +
'''Aim of the experiment:''' Introducing RFC25 pre- and suffix into parts of P73, P80 and P81.
 +
 +
'''Operational sequence:'''
 +
 +
* '''PCR reaction mixture'''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|10&nbsp;µl
 +
|5x OneTaq Standard Reaction Buffer
 +
|-
 +
|1&nbsp;µl
 +
|10&nbsp;mM dNTPs
 +
|-
 +
|1&nbsp;µl
 +
|10&nbsp;µM Forward Primer (For P73: O46 (TUM12-PhyBGal4bd-fw); for P80: O31 (TUM12-LSPS-fw); for P81: O50 (TUM12-Phyb-fw))
 +
|-
 +
|1&nbsp;µl
 +
|10&nbsp;µM Reverse Primer (For P73: O47 (TUM12-PhyBGal4bd-rv); for P80: O32 (TUM12-LSPS-rv ); for P81: O51 (TUM12-Phyb-rv))
 +
|-
 +
|0.25&nbsp;µL
 +
|OneTaq Hot Start DNA Polymerase (Finally: 1.25&nbsp;units/50&nbsp;µL)
 +
|-
 +
|1&nbsp;µl
 +
|Plasmid DNA (P80 or P73 or P81)
 +
|-
 +
|35.75&nbsp;µL
 +
|ELGA Water
 +
|-
 +
|=50&nbsp;µL
 +
|'''TOTAL'''
 +
|}
 +
 +
* The '''gradient''' PCR program was performed after following scheme with following conditions (Tm=58&nbsp; &Delta;G=5&nbsp;°C; P73 in row 9(=61.1&nbsp;°C); P80 in row 1 (=53.0&nbsp;°C); P81 in row 7(=58.4&nbsp;°C):
 +
{|cellspacing="0" border="1"
 +
|Initial denaturation
 +
|94&nbsp;°C
 +
|30&nbsp;s
 +
|-
 +
|30 cycles
 +
|94&nbsp;°C
 +
|30&nbsp;s
 +
|-
 +
|
 +
|Tm=58&nbsp;°C; &Delta;G=5&nbsp;°C
 +
|150&nbsp;s
 +
|-
 +
|
 +
|68&nbsp;°C
 +
|60&nbsp;s
 +
|-
 +
|Final extension
 +
|68&nbsp;°C
 +
|5&nbsp;min
 +
|-
 +
|Hold
 +
|4&nbsp;°C
 +
|overnight
 +
|}
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Miniprep, analcytical digestion and gelelectrophoresis of pGADT7 and pGBKT7 ===
 +
 +
'''Investigator:''' Jeff, Saskia
 +
 +
'''Aim of the experiment:'''Miniprep, analcytical digestion and gelelectrophoresis of pGADT7 (tube P98, EcoRI and BamHI)and pGBKT7 (tube P99, BamHI and PstI)
 +
 +
'''Operational sequence:'''
 +
 +
* Operated after standard protocol of the lab for analytical digestion and gelelectrophoresis.
 +
 +
* Gel '''OK''', like expected
 +
 +
[[File:TUM12_20120713.jpg|500px|Analytical digestion and gelelectrophoresis of pGADT7 (EcoRI and BamHI)and pGBKT7 (EcoRI and PstI).]]
 +
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Preperative digestion and gelelectrophoresis of P79 and O56 ===
 +
'''Investigator:''' Jeff, Saskia, Georg
 +
 +
'''Aim of the experiment:''' Preperativ gelelectrophoresis and gel of digested PCR product of LexA (NgoMIV+PstI) and pSB1C3 containing the RFC25 compatible 20aa linker with RFC25 pre- and suffix (AgeI and PstI).
 +
 +
'''Operational sequence:'''
 +
 +
* Operated after standard protocol of the lab for preperative digestion and gelelectrophoresis.
 +
 +
* Gel '''OK''', like expected.
 +
 +
[[File:TUM12_20120713_prep_gel.jpg|500px|Preperative digestion and gelelectrophoresis of digested PCR product of LexA (NgoMIV+PstI) and pSB1C3 containing the RFC25 compatible 20aa linker with RFC25 pre- and suffix  (AgeI and PstI)??? Digestion with AgeI and PstI.]]
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Preparative digest of P19===
 +
'''Investigator:Daniela, Ingmar'''
 +
 +
'''Digestion of P19 with ApaI'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|20 µl
 +
|P19 (concentration: 53.8 ng/µl)
 +
|-
 +
|4 µl
 +
|Buffer B
 +
|-
 +
|2 µl
 +
|ApaI (10 U/µl)
 +
|-
 +
|14 µl
 +
|ddH2O
 +
|}
 +
 +
Incubation: 37 °C, 3 h
 +
 +
'''DNA preparative gel electrophoresis'''
 +
*gel: 1% with LMP-agarose
 +
*band 1: P50 digested with XbaI and PstI
 +
*band 2: P50 digested with XbaI and NgOMIV
 +
*70 V, 90 min
 +
 +
'''Gelextration'''
 +
*cut the bands
 +
*QIAquick Gel Extractrion Kit was used
 +
** step 6 was left out
 +
** step 9: 30µl buffer, 4 min incubation
 +
</div>
 +
 +
<div class="coumaryl">
 +
===  Gradient PCR of PAL to optimize the primer annealing temperature  ===
 +
 +
'''Investigator: Ingmar'''
 +
 +
Aim of the experiment: As all PCR experiments to amplify the PAL gene contained in P19 failed, we decided to run a gradient PCR to optimize the primer annealing temperature.
 +
 +
'''PCR'''<br>
 +
'''Reaction batch '''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1 µl
 +
|10x Pfu Ultra II buffer
 +
|-
 +
|0.5 µl
 +
|Plasmid P19 template
 +
|-
 +
|0.5 µl
 +
|1:10 dilution of O15 (10 pmol/µL)
 +
|-
 +
|0.5 µl
 +
|1:10 dilution of O59 (10 pmol/µL)
 +
|-
 +
|0.25 µl
 +
|dNTP mix
 +
|-
 +
|7 µl
 +
|ddH2O
 +
|-
 +
|0.25 µl
 +
|Pfu Ultra II DNA polymerase (2.5 U / µl)
 +
|}
 +
 +
'''PCR cycling parameters'''
 +
{|cellspacing="0" border="1"
 +
|'''Segment'''
 +
|'''Cycles'''
 +
|'''Temperature'''
 +
|'''Time'''
 +
|-
 +
|1
 +
|1
 +
|95 °C
 +
| 30 sec
 +
|-
 +
|2
 +
|29
 +
|95°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|gadient 33-47.5 °C
 +
| 1 min
 +
|-
 +
|
 +
|
 +
|68°C
 +
| 3 min
 +
|-
 +
|3
 +
|1
 +
|68°C
 +
|5 min
 +
|-
 +
|4
 +
|1
 +
|4°C
 +
|infinity
 +
|-
 +
|}
 +
 +
*Verification of the PCR by agarose gel electrophoresis:
 +
 +
10 µl of each PCR tube was mixed with 2 µl 6x DNA loading buffer and loaded into the gel. The separation process lasted 1h at 90 V in a 1% agarose gel.
 +
 +
[[File:TUM12 gradient PCR PAL 13.07.2012.jpg|500px|Gel picture of gradient PCR of P19]]
 +
*A bond at the expected length of 2160 bp appears at 47 °C. Therefore a PCR using this primer annealing temperature will done on sunday.
 +
 +
</div>
 +
<div class="coumaryl">
 +
 +
===Transformation of P94 - P97 (ligationproducts of pSB1C3 with CHS and OMT respectively) in ''E.coli''===
 +
Investigator: Daniela
 +
* adding 5µl ligation product (P94-P97) in 100µl competent XL blue ''E.coli'' cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (Withouth antibiotica) and incubate at 37°C, 30 min, 180 rpm
 +
* plate on agar with Ampicillin over night
 +
 +
wrong antibiotica was used!!! Repetition follows!!!
 +
Nevertheless, some colonies could be observed.
 +
</div>
 +
 +
<div class="limonene">
 +
 +
===Ligation of LIMS in pYESnew and pSB1C3===
 +
'''Investigator:Andrea'''
 +
 +
'''Transformation'''
 +
* adding 5µl ligation product (PCR1-PCR7) in 100µl competent XL blue ''E.coli'' cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 30 min, 180 rpm
 +
* plate on agar with Ampicillin (pYESnew) or Chloramphenicol (pSB1C3) over night
 +
</div>
 +
 +
<div class="thaumatin">
 +
 +
=== Preparation of YPD medium ===
 +
 +
'''Investigator: A lot of Alois, Martin'''
 +
 +
'''Manual for YPD production'''
 +
Yeast Extract Peptone Dextrose Medium (1 liter)
 +
*1% yeast extract
 +
*2% peptone
 +
*2% dextrose (D-glucose)
 +
 +
1. Dissolve the following in 1000 ml of water:
 +
*10 g yeast extract
 +
*20 g peptone
 +
*20 g dextrose (see note below if making plates)
 +
 +
2. Autoclave for 20 minutes on liquid cycle.
 +
 +
3. Store medium at room temperature. The shelf life is approximately one to two months.
 +
 +
</div>
 +
 +
== '''Sunday, July 15th ''' ==
 +
 +
<div class="light_switchable_promoter">
 +
=== PCR purification of PCR products of P73, P80 and P81 and analytical gelelectrophoresis ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the experiment:''' PCR was performed to introduce restriction sites to the gene of interest. MfeI and BamHI for P73 and RFC25 pre- and suffix for P80 and P81. The aim is to contruct fusion proteins with the aid of these restriction sites.
 +
 +
'''Operational sequence:'''
 +
 +
* PCR cleaning with Qiagen PCR purification kit after manufacturer's protocol.
 +
 +
* Analytical gelelectrophoresis (1% agarose) at 90&nbsp; for 60&min.
 +
 +
[[File:TUM12_20120715.jpg|500px|PCR of P73, P80 and P81. As expected the only working PCR is P80 because the miniprep of P73 and P81 from Havard university are already corrupt!]]
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===  PCR of PAL consless using the optimized primer annealing temperature of 47 °C ===
 +
 +
'''Investigator: Ingmar'''
 +
 +
Aim of the experiment: Amplification of the PAL gene contained in P19.
 +
 +
'''PCR'''<br>
 +
'''Reaction batch '''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5 µl
 +
|10x Pfu Ultra II buffer
 +
|-
 +
|2.5 µl
 +
|Plasmid P19 template
 +
|-
 +
|2.5 µl
 +
|1:10 dilution of O15 (10 pmol/µL)
 +
|-
 +
|2.5 µl
 +
|1:10 dilution of O59 (10 pmol/µL)
 +
|-
 +
|1.25 µl
 +
|dNTP mix
 +
|-
 +
|35 µl
 +
|ddH2O
 +
|-
 +
|1.25 µl
 +
|Pfu Ultra II DNA polymerase (2.5 U / µl)
 +
|}
 +
 +
'''PCR cycling parameters'''
 +
{|cellspacing="0" border="1"
 +
|'''Segment'''
 +
|'''Cycles'''
 +
|'''Temperature'''
 +
|'''Time'''
 +
|-
 +
|1
 +
|1
 +
|95 °C
 +
| 30 sec
 +
|-
 +
|2
 +
|29
 +
|95°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|47 °C
 +
| 1 min
 +
|-
 +
|
 +
|
 +
|68°C
 +
| 3 min
 +
|-
 +
|3
 +
|1
 +
|68°C
 +
|5 min
 +
|-
 +
|4
 +
|1
 +
|4°C
 +
|infinity
 +
|-
 +
|}
 +
 +
PCR successful - Band at about 2,1 kb :
 +
 +
[[File:TUM12_120716_PCR_PALconsless.jpg|800px|Analytical gelelectrophoresis of PCR product of PAL]]
 +
 +
</div>
 +
</div>
 +
 +
=Week 6=
 +
<!--
 +
<p class="vector_design">'''Vector Design (2 Experiments):'''</p>
 +
*
 +
 +
<p class="limonene">'''Limonene (6 Experiments):'''</p>
 +
*
 +
 +
<p class="coumaryl">'''Xanthohumol (17 Experiments):'''</p>
 +
*
 +
 +
<p class="thaumatin">'''Thaumatin (3 Experiments):'''</p>
 +
* Inoculation of YPD medium with S. cerevisiae and brewing yeast strain 34/70
 +
 +
<p class="constitutive_promoter">'''Constitutive Promoter (6 Experiments):'''</p>
 +
*
 +
 +
<p class="light_switchable_promoter">'''Light Switchable Promoter (16 Experiments):'''</p>
 +
*
 +
 +
<html><a class="WDetails" href="#Week_6" id="Week_6">Show Details ...</a></html>
 +
-->
 +
<div class="week" id="WWeek_6">
 +
== '''Monday, July 16th ''' ==
 +
<div class="constitutive_promoter">
 +
===Inoculation of ADH1-T (iGEM)===
 +
 +
'''Investigator:''' Georg
 +
 +
* Transformantion with ADH1-T didn't go well
 +
* By iGEM transformed cells with ADH1-T were inoculated onto Amp-LB-Plates
 +
* Cells were to grow at room-temperature over the weekend
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
=== Preparative digestion and gelelectrophoresis of P86 and P55 ===
 +
'''Investigator:'''Georg
 +
'''Aim of the experiment:''' Digestion of CYC1-Terminator, ADH1-Promoter for ligation '''
 +
 +
* Preparative digestion after manufacturer's advice (NEB) with 20 u PstI and 20 u XbaI in 1x Tango buffer with 25 µl DNA and 4 µl NEB4 10x buffer and 0,4 µl 100x BSA. Water was added to a volume of 40 µl. Restriction time was 3 hours at 37 °C; 3 hours.
 +
 +
* Preparative gelelectrophoresis after laboratory's standart protocol. (70&nbsp;V, 90&nbsp;min)
 +
 +
[[File:Preparative digestion ADH1-p, CYC1-t.png]]
 +
 +
* Gel was stored at -20 °C.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Preperative digestion and gelelectrophoresis of P98 and PCR26 ===
 +
 +
'''Investigator:''' Jeff, Georg
 +
 +
'''Aim of the experiment:''' Construction of Gal4AD-Pif3, a part of the light-switchable promoter system.
 +
 +
'''Operational sequence:'''
 +
 +
* Preperative digestion after manufacturer's (Fermentas) advise for double-digestion for EcoRI+BamHI and MfeI(MunI)+BamHI; EcoRI+BamHI: Buffer 2X Tango™, 2-fold excess of BamHI; MfeI(MunI)+BamHI: Buffer G.
 +
 +
* Preperative gelelectrohphoresis after laboratory's standard protocol. (70&nbsp;V, 90&nbsp;min)
 +
 +
[[File:TUM12_20120716_prep_gel.jpg|500px]]
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Plating of received ''E.&nbsp;coli'' containing biobrick BBa_K165055 ===
 +
'''Investigator: Jeff'''
 +
 +
'''Aim of the experiment:''' The received biobrick BBa_K165055 (LexA binding sites + mCYC + Kozak + YFPx2 + ADH1 terminator) were already transformed in ''E.&nbsp;coli'' and were in an agar stabs. These ''E.&nbsp;coli'' cells were transferred with an inoculation loop on antibiotic selection plates and were incubated over night.
 +
 +
'''Operational sequence:'''
 +
* Bacterias containing plasmids with biobricks were transferred with a sterile inoculation loop on antibiotic plates and were incubated at 37&nbsp;°C overnight.
 +
 +
* The biobricks was BBa_K165055 in BBa_J63009 plasmid (AmpR, low copy plasmid)
 +
</div>
 +
 +
<div class="thaumatin">
 +
 +
=== Preparatory culture of ''S.&nbsp;cerevisiae'' ===
 +
 +
'''Investigator:''' Alois, Martin
 +
 +
'''Aim of the experiment:''' In order to have sufficient viable cells to inoculate an experimental culture in which we want to compare the growth rate of ''s. cerevisiae'' with a strain of brewing yeast (34/70 ''s. pastorianus weihenstephan'') we aim to establish a preparatory over night culture.
 +
 +
'''Operational sequence:'''
 +
A sample of -80°C stored ''s. cerevisiae'' (glycerol stock) is used to inoculate 20ml of YPD-medium. This is shaken overnight at 30°C.
 +
</div>
 +
 +
<div class="limonene">
 +
=== Miniprep of plasmids containing lavendula limonene synthase/citrus limonene synthase ===
 +
 +
'''Investigator:''' Lara
 +
 +
'''Aim of the experiment:''' Extract plasmids (pYESnew and pBS1C3) that contain limonene synthase/citrus limonene synthase.
 +
 +
Miniprep with Qiagen Kit; Plasmids p116-p122. Restriction digest with Xba1 and Pst1.
 +
 +
Plasmid DNA concentrations:
 +
 +
p116: 285 ng/µl
 +
 +
p117: 337 ng/µl
 +
 +
p118: 520 ng/µl
 +
 +
p119: 370 ng/µl
 +
 +
p120: 320 ng/µl
 +
 +
p121: 320 ng/µl
 +
 +
p122: 335 ng/µl
 +
 +
</div>
 +
 +
<div class="limonene">
 +
 +
=== Restriction digest of p116-p122 ===
 +
 +
'''Investigator:''' Lara
 +
 +
'''Aim of the experiment:''' Analytical restriction digest of plasmids p116, p117, p118, p119, p120, p121, p122.
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2 µl
 +
|DNA
 +
|-
 +
|2 µl
 +
|Buffer 4
 +
|-
 +
|0,25 µl
 +
|Xba1
 +
|-
 +
|0,25 µl
 +
|Pst1-HF
 +
|-
 +
|15,3 µl
 +
|ddH2O
 +
|}
 +
 +
Incubation: 37 °C; 1,5 h
 +
 +
Restriction digest with Xba1 and Pst1-HF.
 +
 +
</div>
 +
 +
<div class="limonene">
 +
 +
=== Analytical gel electrophoresis of p116-p122 ===
 +
 +
'''Investigator:''' Lara
 +
 +
'''Aim of the experiment:''' Analytical gel electrophoresis of products from restriction digest of plasmids p116, p117, p118, p119, p120, p121, p122.
 +
 +
p116: PCR1/pYES
 +
 +
p117: PCR2/pYES
 +
 +
p118: PCR4/pYES
 +
 +
p119: PCR4/pSB1C3
 +
 +
p120: PCR5/pYES
 +
 +
p121: PCR6/pYES
 +
 +
p122: PCR7/pYES
 +
 +
[[File:TUM12_LS_gelelectrophoresis1607.png]]
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
=== Midiprep of pSB1C3 (RFC25-compatible) ===
 +
 +
'''Investigator:'''Mary
 +
 +
'''Aim of the experiment:''' Get a lot of pSB1C3 for further experiments
 +
 +
Midipreparation of the pellet from over night cultures (''E.coli'', Transformed with pSB1C3 - RFC25; name in registry: K365005)
 +
 +
was done with the Midiprep Kit from Qiagen
 +
 +
Result was named as P123 and had the concentration: 469,5 ng/µl (100µl at all)
 +
 +
</div>
 +
 +
<div=Xanthohumol>
 +
 +
<div class="coumaryl">
 +
 +
=== PCR-Products of PAL (consless): digestion, extraction and ligation ===
 +
 +
'''investigator: ''' Daniela, Mary
 +
 +
'''Aim of the experiment:''' digestion of PCR-Products to gain the correct cutting sites for ligation in pYES2 and pSB1C3 afterwards
 +
 +
Purification of PCR-Products with PCR-Purification Kit
 +
 +
*digestion with Xba1 and HF-Age1 (both NEB)
 +
 +
{| class="wikitable" cellpadding="10" border=1px
 +
| '''volume''' || '''reagent'''
 +
|-
 +
| 25µl || PCR-product
 +
|-
 +
| 5µl || Buffer NEB4
 +
|-
 +
| 0.5µl || BSA
 +
|-
 +
| 1µl || Xba1 (NEB; 20u/µl)
 +
|-
 +
| 1µl || HF-Age1 (NEB; 20u/µl) 
 +
|-
 +
| 17.5µl || bidest. sterile H2O
 +
|}
 +
 +
digestion took 3h at 37°C
 +
 +
*extraction from preparative gel:
 +
 +
[[File:TUM_12_PAL_consless_(digested_with_Xba_Age).png|400px]]
 +
 +
</div>
 +
<div class="coumaryl">
 +
 +
===  PCR of PAL+cons using the optimized primer annealing temperature of 47 °C ===
 +
 +
'''Investigator: Daniela'''
 +
 +
Aim of the experiment: Amplification of the PAL cons gene contained in P19.
 +
 +
'''PCR'''<br>
 +
'''Reaction batch '''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5 µl
 +
|10x Pfu Ultra II buffer
 +
|-
 +
|2.5 µl
 +
|Plasmid P19 template
 +
|-
 +
|2.5 µl
 +
|1:10 dilution of O22 (10 pmol/µL)
 +
|-
 +
|2.5 µl
 +
|1:10 dilution of O59 (10 pmol/µL)
 +
|-
 +
|1.25 µl
 +
|dNTP mix
 +
|-
 +
|35 µl
 +
|ddH2O
 +
|-
 +
|1.25 µl
 +
|Pfu Ultra II DNA polymerase (2.5 U / µl)
 +
|}
 +
 +
'''PCR cycling parameters'''
 +
{|cellspacing="0" border="1"
 +
|'''Segment'''
 +
|'''Cycles'''
 +
|'''Temperature'''
 +
|'''Time'''
 +
|-
 +
|1
 +
|1
 +
|95 °C
 +
| 30 sec
 +
|-
 +
|2
 +
|29
 +
|95°C
 +
| 30 sec
 +
|-
 +
|
 +
|
 +
|47 °C
 +
| 1 min
 +
|-
 +
|
 +
|
 +
|68°C
 +
| 3 min
 +
|-
 +
|3
 +
|1
 +
|68°C
 +
|5 min
 +
|-
 +
|4
 +
|1
 +
|4°C
 +
|infinity
 +
|-
 +
|}
 +
 +
PCR was succesful, band at 2,1kb:
 +
 +
[[File:TUM12_120717_PCR_PALconsens.jpg|500px]]
 +
 +
</div>
 +
<div class="coumaryl">
 +
 +
===Transformation of P94 - P97 (ligationproducts of pSB1C3 with CHS and OMT respectively) in ''E.coli''===
 +
Investigator: Daniela
 +
* adding 5µl ligation product (P94-P97) in 100µl competent XL blue E.coli cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 45 min, 180 rpm
 +
* plate on agar with chloramphenicol over night
 +
-> were succesfull: some colonies were grown
 +
</div>
 +
 +
== '''Tuesday, July 17th ''' ==
 +
<div class="constitutive_promoter">
 +
===Picking of ADH1-T colonies===
 +
 +
'''Investigator:''' Georg
 +
 +
* To 5 µl LB-Medium,5 µl of 1000x Amp were added
 +
* 5 colonies from the plate with ADH1-P were picked
 +
* Each colony was transferred into 5 ml LB-Medium with Chloramphenicol
 +
* Incubation over night at 37°C in the 180rpm cell-culture shaker.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Picking of transformed ''E.&nbsp;coli'' containing biobrick BBa_K165055 ===
 +
</div>
 +
 +
<div class="coumaryl">
 +
===Preparative digest of P123 (pSB1C3 RFC25)===
 +
'''Investigator: Mary, Daniela'''
 +
 +
'''Digestion of P123 with Xbal/PstI and XbaI/AgeI
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|23.1 µl
 +
|ddH2O
 +
|-
 +
|4 µl
 +
|NEB4
 +
|-
 +
|0.4 µl
 +
|10x BSA
 +
|-
 +
|1 µl
 +
|XbaI (20 U/µl)
 +
|-
 +
|1.5 µl
 +
|PstI (10U/µl)
 +
|-
 +
|10 µl
 +
|P123
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|23.35 µl
 +
|ddH2O
 +
|-
 +
|4 µl
 +
|NEB4
 +
|-
 +
|0.4 µl
 +
|10x BSA
 +
|-
 +
|1 µl
 +
|XbaI (20 U/µl)
 +
|-
 +
|1.25 µl
 +
|AgeI (10U/µl)
 +
|-
 +
|10 µl
 +
|P123
 +
|}
 +
 +
Incubation: 37 °C, 3 h
 +
 +
'''DNA preparative gel electrophoresis'''
 +
*gel: 1% with LMP-agarose
 +
*band 1: P123 digested with XbaI and PstI
 +
*band 2: P123 digested with XbaI and AgeI
 +
*70 V, 90 min
 +
 +
'''Gelextration'''
 +
*cut the bands
 +
*QIAquick Gel Extractrion Kit was used
 +
** step 6 was left out
 +
** step 9: 30µl buffer, 4 min incubation
 +
 +
Products were names as follows:
 +
* P123 digested with XbaI and PstI: P132
 +
* P123 digested with XbaI and AgeI: P133
 +
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
=== PCR-Products of PAL (with consensussequence): digestion and extraction ===
 +
 +
'''investigator: ''' Daniela, Mary
 +
 +
'''Aim of the experiment:''' digestion of PCR-Products to gain the correct cutting sites for ligation in pTUM104 and pSB1C3 afterwards
 +
 +
Purification of PCR-Products with PCR-Purification Kit
 +
 +
*digestion with Xba1 and HF-Age1 (both NEB)
 +
 +
{| class="wikitable" cellpadding="10" border=1px
 +
| '''volume''' || '''reagent'''
 +
|-
 +
| 25µl || PCR-product
 +
|-
 +
| 5µl || Buffer NEB4
 +
|-
 +
| 0.5µl || BSA
 +
|-
 +
| 1µl || Xba1 (NEB; 20u/µl)
 +
|-
 +
| 1µl || HF-Age1 (NEB; 20u/µl) 
 +
|-
 +
| 17.5µl || bidest. sterile H2O
 +
|}
 +
 +
digestion took 3h at 37°C
 +
 +
*extraction from preparative gel:
 +
 +
[[File:TUM12_120717_PAL+_prepGel.jpg|800px]]
 +
 +
</div>
 +
 +
<div class="coumaryl">
 +
=== Picking Clones of CHS and OMT in pSB1C3-RFC25 ===
 +
 +
'''investigator: ''' Daniela, Mary
 +
 +
'''Aim of the experiment:''' preculture over night for the miniprep next day
 +
 +
</div>
 +
<div class="coumaryl">
 +
 +
=== Repetition of ligation of PCR 15-20 (4CL, CHS and OMT) with pTUM104 (P71 and P72 respectively)===
 +
 +
'''Investigator: Mary, Daniela'''
 +
 +
Concentration (Nano Drop: <br />
 +
4CL+ (PCR 15) = 40.3 ng/µl<br />
 +
4CL- (PCR 16) = 37.3 ng/µl<br />
 +
CHS+ (PCR 17) = 46.1 ng/µl<br />
 +
CHS- (PCR 18) = 51.2 ng/µl<br />
 +
OMT+ (PCR 19) = 22.3 ng/µl<br />
 +
OMT- (PCR 20) = 16.7 ng/µl<br />
 +
<br />
 +
P50 digested with XbaI and PstI = 23.9 ng/µl (the digested Plasmid has the number P71)<br />
 +
P50 digested with XbaI and NgOMIV = 28.4 ng/µl (the digested Plasmid has the number P72)<br />
 +
<br />
 +
*required volumes were calculated using Lab Tools
 +
4CL+ (PCR 15) with P71
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5.27 µl
 +
|P71
 +
|-
 +
|2.73 µl
 +
|4CL+ (PCR 15)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
 +
4CL- (PCR 16) with P71
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5.13 µl
 +
|P71
 +
|-
 +
|2.87 µl
 +
|4CL- (PCR 16)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
CHS+ (PCR 17) with P72
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5.84 µl
 +
|P72
 +
|-
 +
|2.16 µl
 +
|CHS+ (PCR 17)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
CHS- (PCR 18) with P72
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|6 µl
 +
|P72
 +
|-
 +
|2 µl
 +
|CHS- (PCR 18)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
OMT+ (PCR 19) with P72
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4.45 µl
 +
|P72
 +
|-
 +
|3.55 µl
 +
|OMT+ (PCR 19)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
OMT- (PCR 20) with P72
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|3.87 µl
 +
|P72
 +
|-
 +
|4,13 µl
 +
|OMT- (PCR 20)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
Negative control
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5 µl
 +
|P71 or P72
 +
|-
 +
|3 µl
 +
|ddH2O
 +
|-
 +
|1 µl
 +
|T4 DNA-ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
 +
*water bath 16 °C
 +
 +
</div>
 +
<div class="coumaryl">
 +
===Transformation of ligationproducts of 4CL, OMT and CHS respectively in pTUM104 in ''E.coli''===
 +
 +
Investigator: Mary,Daniela
 +
* adding 5µl ligation product  in 100µl competent XL blue E.coli cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 45 min, 180 rpm
 +
* plate on agar with ampicillin over night
 +
 +
results:
 +
 +
*CHS+ in pTUM104 (P72) 100 µl: 7 clones
 +
*CHS- in pTUM104 (P72) 100 µl: 9 clones
 +
*CHS+ in pTUM104 (P72) Pellet: 111 clones
 +
*CHS- in pTUM104 (P72) Pellet: 77 clones
 +
 +
*4CL+ in pTUM104 (P71) 100 µl: 9 clones
 +
*4CL- in pTUM104 (P71) 100 µl: 3 clones
 +
*4CL+ in pTUM104 (P71) Pellet: 47 clones
 +
*4CL- in pTUM104 (P71) Pellet: 45 clones
 +
 +
*OMT+ in pTUM104 (P72) 100 µl: 7 clones
 +
*OMT- in pTUM104 (P72) 100 µl: 5 clones
 +
*OMT+ in pTUM104 (P72) Pellet: 30 clones
 +
*OMT- in pTUM104 (P72) Pellet: 53 clones
 +
 +
*negative control P71 100 µl: 14
 +
*negative control P71 Pellet: 71
 +
*negative control P72 100 µl: 7
 +
*negative control P72 Pellet: 64
 +
</div>
 +
<div class="thaumatin">
 +
 +
=== Inoculation of YPD medium with ''S. cerevisiae'' and brewing yeast strain 34/70 (part 1/3) ===
 +
 +
'''investigator: ''' Maddin, Aloisius
 +
 +
'''Aim of the experiment:''' Discrimination of growing ability in wort medium
 +
 +
4 different worts:
 +
*21%, without hop
 +
*21%, without hop, autoclaved
 +
*16%, with hop
 +
*16%, with hop, autoclaved
 +
 +
were diluted with ELGA to a solution of 12%. These 4 different worts were inoculated with 5ml of preparatory culture of ''S. cerevisiae'' and 1ml of brewing yeast strain 34/70 (provided by the Forschungsbrauerei, Weihenstephan). Start OD was measured at 550nm (for table see next experiment).
 +
Those 8 flasks were incubated over night at room temperature and 130 rpm.
 +
</div>
 +
 +
== '''Wednesday, July 18th''' ==
 +
<div class="constitutive_promoter">
 +
=== Minipreparation of ADH1-T Plasmids===
 +
 +
'''Investigator:''' Georg
 +
* ADH1-T Plasmids were extracted according to protocoll of Quiaprep gelextraction protocoll
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
 +
=== Analytical digestion of ADH1-T and gelelectrophoresis===
 +
'''Investigator:''' Georg
 +
* Reaction batch for digestion:
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1,25 µl
 +
|PstI-HF (NEB)
 +
|-
 +
|1,25 µl 
 +
|XbaI (NEB)
 +
|-
 +
|5 µl
 +
|NEB-4 buffer
 +
|-
 +
|0,5 µl
 +
|100 x BSA (NEB)
 +
|-
 +
|79,5 µl
 +
|dd H20
 +
|-
 +
|= 87,5µl
 +
|'''TOTAL'''
 +
|}
 +
 +
* 17,5 µl Mastermix were added to 2,5 µl plasid DNA
 +
 +
[[File:20120718  BBa_j63002.PNG]]
 +
 +
* They have sent the wrong insert
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Ligation of plasmid pSB1C3 containing 20aaLinker with LexA and plasmid containing Gal4AD with Pif3 for fusion protein construction<br> ===
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Miniprep of overnight culture from picked transformed ''E. coli'' containing biobrick BBa_K165055 ===
 +
</div>
 +
 +
<div class="thaumatin">
 +
 +
=== Inoculation of YPD medium with ''S. cerevisiae'' and brewing yeast strain 34/70 (part 2/3) ===
 +
 +
'''Investigators: ''' Martin, Alois
 +
 +
'''Aim of the experiment:''' Discrimination of growing ability in wort medium.
 +
 +
After 12h of cultivating the optical density (OD) at 550nm was determined:
 +
 +
{|cellspacing="0" border="2"
 +
|'''Assay'''
 +
|'''Yeast'''
 +
|'''Autoclaved'''
 +
|'''Hop'''
 +
|'''OD 0h'''
 +
|'''OD 12h'''
 +
|'''delta OD'''
 +
|-
 +
|A1
 +
|''S. cerevisae''
 +
|No
 +
|No
 +
|1.2
 +
|2.2
 +
| + 1.0
 +
|-
 +
|A2
 +
|''S. cerevisae''
 +
|'''Yes'''
 +
|No
 +
|2.0
 +
|2.6
 +
| + 0.6
 +
|-
 +
|A3
 +
|''S. cerevisae''
 +
|No
 +
|'''Yes'''
 +
|1.8
 +
|2.4
 +
| + 0.6
 +
|-
 +
|A4
 +
|''S. cerevisae''
 +
|'''Yes'''
 +
|'''Yes'''
 +
|2.4
 +
|2.7
 +
| + 0.3
 +
|-
 +
|B1
 +
|34/70
 +
|No
 +
|No
 +
|1.6
 +
|2.5
 +
| + 0.9
 +
|-
 +
|B2
 +
|34/70
 +
|'''Yes'''
 +
|No
 +
|3.0
 +
|2.9
 +
| '''- 0.1'''
 +
|-
 +
|B3
 +
|34/70
 +
|No
 +
|'''Yes'''
 +
|2.2
 +
|2.5
 +
| + 0.3
 +
|-
 +
|B4
 +
|34/70
 +
|'''Yes'''
 +
|'''Yes'''
 +
|2.7
 +
|2.8
 +
| + 0.1
 +
|}
 +
 +
After 12h of cultivating the ''S. cerevisiae'' is growing quite well. There is a tendency that the brewing yeast strain 34/70 is not capable to grow sufficiently in autoclaved (e.g. proteinfree) medium. Since the 34/70 was stored at 4°C over 5 days, directly used to inoculate the medium (no preparatory culture) and we only used 1ml to inoculate with (compared to 5ml of the preparatory culture of ''S. cerevisiae'' - because of obvious differences in opacity of the inoculation media) there is not yet a conclusion to be drawn. We decided to incubate for another 24h at least.
 +
</div>
 +
 +
<div class="vector_design">
 +
=== New Miniprep of P50 pTUM104 ===
 +
 +
'''Investigators: Andrea'''
 +
 +
NanoDrop concentration: 303.6 ng/µl (260/280: 1.89)
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Transformation of P50 (pTUM104) in ''E.coli''===
 +
Investigator: Katrin
 +
 +
'''Aim of the experiment:''' Get more P50(pTUM104) for further experiments
 +
* adding 2-3 µl of plasmid P50 in 100µl competent XL blue E.coli cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (withouth antibiotica) and incubate at 37°C, 30 min, 180 rpm
 +
* plate on agar with Ampicillin over night
 +
</div>
 +
 +
<div class="coumaryl">
 +
===Ligation of 4CL and PAL in pSB1C3-RFC25 and ligation of PAL in pTUM104===
 +
Investigator: Katrin, Daniela
 +
 +
Concentration (Nano Drop: <br />
 +
4CL+ (PCR 15) = 40.3 ng/µl<br />
 +
4CL- (PCR 16) = 37.3 ng/µl<br />
 +
PAL+ (PCR 32) = 25 ng/µl<br />
 +
PAL- (PCR 33) = 16.8 ng/µl<br />
 +
<br />
 +
P132 (pSB1C3-RFC25 digested with Xbal and Pstl) = 33.5 ng/µl<br />
 +
P133 (pSB1C3-RFC25 digested with Xbal and AgeI) = 41.8 ng/µl<br />
 +
<br />
 +
*required volumes were calculated using Lab Tools
 +
4CL+ (PCR 15) with P132 (pSB1C3-RFC25)
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.64 µl
 +
|P132
 +
|-
 +
|5.36 µl
 +
|4CL+ (PCR 15)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|2 µl
 +
|T4-ligase buffer (10x)
 +
|-
 +
|9 µl
 +
|ddH2O
 +
|}
 +
 +
4CL- (PCR 16) with P132 (pSB1C3-RFC25)
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.51 µl
 +
|P132
 +
|-
 +
|5.49 µl
 +
|4CL- (PCR 16)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|2 µl
 +
|T4-ligase buffer (10x)
 +
|-
 +
|9 µl
 +
|ddH2O
 +
|}
 +
 +
PAL+ (PCR 32) with P133 (pSB1C3-RFC25)
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.29 µl
 +
|P133
 +
|-
 +
|6.71 µl
 +
|PAL+ (PCR 32)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|2 µl
 +
|T4-ligase buffer (10x)
 +
|-
 +
|9 µl
 +
|ddH2O
 +
|}
 +
 +
PAL- (PCR 33) with P133 (pSB1C3-RFC25)
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|0.91 µl
 +
|P133
 +
|-
 +
|7.09 µl
 +
|PAL- (PCR 33)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|2 µl
 +
|T4-ligase buffer (10x)
 +
|-
 +
|9 µl
 +
|ddH2O
 +
|}
 +
 +
PAL+ (PCR 32) with P72 (pTUM104)
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|3.55 µl
 +
|P72
 +
|-
 +
|4.45 µl
 +
|PAL+ (PCR 32)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|2 µl
 +
|T4-ligase buffer (10x)
 +
|-
 +
|9 µl
 +
|ddH2O
 +
|}
 +
 +
PAL- (PCR 33) with P72 (pTUM104)
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.79 µl
 +
|P72
 +
|-
 +
|5.21 µl
 +
|PAL- (PCR 33)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|2 µl
 +
|T4-ligase buffer (10x)
 +
|-
 +
|9 µl
 +
|ddH2O
 +
|}
 +
Negative control:
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5 µl
 +
|P72, P132 or P133
 +
|-
 +
|12 µl
 +
|ddH2O
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|2 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
*water bath 16 °C
 +
 +
</div>
 +
<div class="coumaryl">
 +
===Miniprep of CHS and OMT in pSB1C3-RFC25===
 +
 +
Investigator: Daniela
 +
 +
'''Aim of the experiment''': Extract plasmids (pSB1C3-RFC25) that contain CHS and OMT
 +
 +
QIAprepS Spin Miniprep Kit
 +
*step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)
 +
the Minipreps were named as follows:
 +
*P135: CHS+ in pSB1C3-RFC25 (c = 150.9 ng/µl)
 +
*P136: CHS- in pSB1C3-RFC25 (c = 204 ng/µl)
 +
*P137: OMT+ in pSB1C3-RFC25 (c = 179.3 ng/µl)
 +
*P138: OMT- in pSB1C3-RFC25 (c = 146.7 ng/µl)
 +
 +
</div>
 +
<div class="coumaryl">
 +
===Control digest of CHS and OMT in pSB1C3-RFC25===
 +
 +
Investigator: Katrin, Daniela
 +
 +
'''Aim of the experiment:''' Test whether the ligation of CHS and OMT in pSB1C3 was successful
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|P135 / P136 / P137 / P138
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|AgeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|0.2 µl
 +
|BSA (100x)
 +
|-
 +
|14.8 µl
 +
|ddH2O
 +
|}
 +
 +
[[File:TUM12 20120718 P135-P138.jpg]]
 +
*pSB1C3: 2070 bp
 +
*CHS: 1173 bp
 +
*OMT: 1059 bp
 +
It seems as if the ligation was successful. However it is strange that the band of OMT is lower that the one of CHS.
 +
we had the wrong information about the length of OMT -> the right length of OMT is 1059 bp, so everything is fine :)
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
=== Picking clones of 4CL, CHS and OMT in pTUM104 ===
 +
 +
'''investigator: ''' Katrin, Daniela
 +
 +
'''Aim of the experiment:''' preculture over night for the miniprep next day
 +
 +
</div>
 +
 +
== '''Thursday, July 19th''' ==
 +
 +
<div class="light_switchable_promoter">
 +
===Transformation of P124+PCR29 and P126+PCR31===
 +
 +
'''Investigator:''' Daniela
 +
 +
'''Aim of the experiment:''' Transformation of the ligation of P124+PCR29 and P126+PCR31 overnight, to see if ligation has been successful.
 +
 +
'''Operational sequence:'''
 +
 +
*P124+PCR29 ->Chlp
 +
*negative control: NK1 ->Chlp
 +
*P126+PCR31 ->Amp
 +
*negative control: NK2 ->Amp
 +
 +
* adding 5µl ligation product in 100 µl competent XL blue E.coli cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 30 min for ampicillin and 45 min for chloramphenicol, 180 rpm
 +
* plate on agar with ampicillin or chloramphenicol over night
 +
 +
'''EDIT from 20.07.2012:''' Unfortunately, no colonies for P124+PCR29 can be identified but colonies on from the concentrated pellet of P126+PCR31 ligation, BUT more but very small colonies on the control plate from the pellet. For further identification the colonies of the ligation has been picked on 21.07.2012 for miniprep and analytical digestion and gelelectrophoresis.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
===Restriction digest of P79===
 +
'''Investigator:''' Roman
 +
 +
'''Aim of the experiment:''' Double digest of p79 with Xba1 and Age1 to prepair it for ligation with an N- terminal nuclear localization signal sequence
 +
 +
40 µl composition
 +
* 18 µl plasmid DNA
 +
* 1 µl Age1
 +
* 1 µl Xba1
 +
* 4 µl NEBuffer 4 (10x)
 +
* 15,6 µl ddH2O
 +
* 0,4 µl BSA (100x)
 +
 
 +
The mixture was incubated over night at 37 °C
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Oligohybridization of single-stranded DNA for creating SV40 nuclear localization signal for fusion proteins for translocating them into the nucleus ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the experiment:''' Oligohybridization of single-stranded DNA (TUM12-SV40-fw and TUM12-SV40-rv)for creating SV40 nuclear localization signal for fusion proteins for translocating them into the nucleus
 +
 +
'''Operational sequence:'''
 +
 +
* 25&nbsp;µL of 100&nbsp;pM of TUM12-SV40-fw and 25&nbsp;µL of 100&nbsp;pM TUM12-SV40-rv in one ERG
 +
 +
* Heating up to 95&nbsp;°C for 30&nbsp;min
 +
 +
* Cooling at RT in a styropor box overnight.
 +
</div>
 +
 +
<div class="thaumatin">
 +
 +
=== Inoculation of YPD medium with ''S. cerevisiae'' and brewing yeast strain 34/70 (part 3/3) ===
 +
 +
'''Investigators: ''' Andrea, (Martin, Alois)
 +
 +
'''Aim of the experiment:''' Discrimination of growing ability in wort medium.
 +
 +
After 40h of cultivating the optical density (OD) at 550nm was determined:
 +
 +
{|cellspacing="0" border="2"
 +
|'''Assay'''
 +
|'''Yeast'''
 +
|'''Autoclaved'''
 +
|'''Hop'''
 +
|'''OD 0h'''
 +
|'''OD 12h'''
 +
|'''OD 40h'''
 +
|'''delta OD'''
 +
|-
 +
|A1
 +
|''S. cerevisae''
 +
|No
 +
|No
 +
|1.2
 +
|2.2
 +
|2.5
 +
| + 1.3
 +
|-
 +
|A2
 +
|''S. cerevisae''
 +
|'''Yes'''
 +
|No
 +
|2.0
 +
|2.6
 +
|2.7
 +
| + 0.7
 +
|-
 +
|A3
 +
|''S. cerevisae''
 +
|No
 +
|'''Yes'''
 +
|1.8
 +
|2.4
 +
|2.6
 +
| + 0.8
 +
|-
 +
|A4
 +
|''S. cerevisae''
 +
|'''Yes'''
 +
|'''Yes'''
 +
|2.4
 +
|2.7
 +
|2.8
 +
| + 0.4
 +
|-
 +
|B1
 +
|34/70
 +
|No
 +
|No
 +
|1.6
 +
|2.5
 +
|2.8
 +
| + 1.2
 +
|-
 +
|B2
 +
|34/70
 +
|'''Yes'''
 +
|No
 +
|3.0
 +
|2.9
 +
|3.0
 +
| '''0'''
 +
|-
 +
|B3
 +
|34/70
 +
|No
 +
|'''Yes'''
 +
|2.2
 +
|2.5
 +
|2.8
 +
| + 0.6
 +
|-
 +
|B4
 +
|34/70
 +
|'''Yes'''
 +
|'''Yes'''
 +
|2.7
 +
|2.8
 +
|2.9
 +
| + 0.2
 +
|}
 +
 +
After 40h of cultivating the ''S. cerevisiae'' is growing comparably to the brewing yeast strain, though we seem to have entered a phase of substrate limitation.
 +
Autoclaved media lack solved protein and is thus for neither of the yeasts a satisfying medium; added hop also represses their growth (''S. cerevisiae'' shows a higher susceptability - which seems logic, since the brewing yeast is selected to survive and prosper in beer brewing environment).
 +
 +
The next step would be to brew with our "laboratory strain" ''S. cerevisiae''.
 +
</div>
 +
 +
<div class="limonene">
 +
 +
===Preparative digest of PCR1 and PCR2 and P50 and P123===
 +
'''Investigator: Andrea'''
 +
 +
'''Digestion of PCR1 and PCR2 with XbaI and AgeI'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|10 µl
 +
|PCR-Product
 +
|-
 +
|2 µl
 +
|NEB Buffer
 +
|-
 +
|0.2 µl
 +
|BSA
 +
|-
 +
|1 µl
 +
|XbaI (10 U/µl)
 +
|-
 +
|1 µl
 +
|AgeI (10 U/µl)
 +
|-
 +
|7 µl
 +
|ddH2O
 +
|}
 +
 +
'''Digestion of P50 with XbaI and NgoMIV'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|10 µl
 +
|P50
 +
|-
 +
|4 µl
 +
|NEB Buffer
 +
|-
 +
|0.4 µl
 +
|BSA
 +
|-
 +
|1 µl
 +
|XbaI (10 U/µl)
 +
|-
 +
|2 µl
 +
|NgoMIV (10 U/µl)
 +
|-
 +
|22.6 µl
 +
|ddH2O
 +
|}
 +
 +
'''Digestion of P123 with XbaI and AgeI'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|20 µl
 +
|P123
 +
|-
 +
|4 µl
 +
|NEB Buffer
 +
|-
 +
|0.4 µl
 +
|BSA
 +
|-
 +
|1 µl
 +
|XbaI (10 U/µl)
 +
|-
 +
|1 µl
 +
|AgeI (10 U/µl)
 +
|-
 +
|14 µl
 +
|ddH2O
 +
|}
 +
 +
Incubation: 37 °C, 3 h
 +
 +
</div>
 +
 +
<div class="limonene">
 +
===Preparative gel of pSB1C3 and pTUM104===
 +
'''Investigator: Andrea'''
 +
 +
*40 µl pSB1C3 + 4 µl loading dye
 +
*40 µl pTUM104 + 4 µl loading dye
 +
*20 µl PCR1 + 2 µl loading dye
 +
 +
*Limonensynthase: 1600 bp
 +
*pTUM104: 5800 bp
 +
*pSB1C3: 2000bp
 +
 +
[[File:19.07.12-prep-gel.png|400px]]
 +
 +
</div>
 +
 +
<div class="limonene">
 +
 +
=== Ligation of digested PCR1 (digested with XbaI and AgeI) with pSB1C3 (digested with XbaI and AgeI)===
 +
 +
'''Investigator: Andrea'''
 +
 +
Concentration (Nano Drop: <br />
 +
LIMS Citrus (PCR 1) = 18.6 ng/µl<br />
 +
P50 (digested with XbaI and NgoMIV) = 24.9 ng/µl<br />
 +
P123 (digested with XbaI and AgeI) = 44.5 ng/µl<br />
 +
 +
<br />
 +
PCR1 with P123 digested with XbaI and AgeI/NgoMIV
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.19µl
 +
|P123
 +
|-
 +
|6.81 µl
 +
|PCR 1
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|1 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
 +
*water bath 16 °C over night
 +
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Miniprep of 4CL, CHS and OMT in pTUM104 (P71 and P72)===
 +
 +
Investigator: Daniela
 +
 +
'''Aim of the experiment''': Extract plasmids (pTUM104) that contain 4CL, CHS and OMT
 +
 +
The day before 2 clones were picked for each enzyme.
 +
 +
QIAprepS Spin Miniprep Kit
 +
*step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)
 +
 +
Concentration:
 +
*4CL+ in pTUM104 clone 1: c = 172.5 ng/µl
 +
*4CL- in pTUM104 clone 1: c = 192.1 ng/µl
 +
*CHS+ in pTUM104 clone 1: c = 136.6 ng/µl
 +
*CHS- in pTUM104 clone 1: c = 85.7 ng/µl
 +
*OMT+ in pTUM104 clone 1: c = 116.0 ng/µl
 +
*OMT- in pTUM104 clone 1: c = 129.7 ng/µl
 +
 +
*4CL+ in pTUM104 clone 2: c = 160.0 ng/µl
 +
*4CL- in pTUM104 clone 2: c = 144.5 ng/µl
 +
*CHS+ in pTUM104 clone 2: c = 128.5 ng/µl
 +
*CHS- in pTUM104 clone 2: c = 116.2 ng/µl
 +
*OMT+ in pTUM104 clone 2: c = 142.8 ng/µl
 +
*OMT- in pTUM104 clone 2: c = 134.5 ng/µl
 +
 +
</div>
 +
<div class="coumaryl">
 +
 +
===Control digest of 4CL, CHS and OMT in pTUM104===
 +
 +
Investigator: Daniela
 +
 +
'''Aim of the experiment:''' Test whether the ligation of 4CL, CHS and OMT in pTUM104 was successful
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|4CL+ in pTUM104 clone 1 / 4CL- in pTUM104 clone 1
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|0.2 µl
 +
|BSA (100x)
 +
|-
 +
|14.8 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|3 µl
 +
|4CL+ in pTUM104 clone 2
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|0.2 µl
 +
|BSA (100x)
 +
|-
 +
|14.3 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|3.5 µl
 +
|4CL- in pTUM104 clone 2
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|0.2 µl
 +
|BSA (100x)
 +
|-
 +
|13.8 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|3.5 µl
 +
|CHS+ in pTUM104 clone 1 / OMT+ in pTUM104 clone 1 / OMT- in pTUM104 clone 1 / CHS+ in pTUM104 clone 2 / OMT+ in pTUM104 clone 2 / OMT- in pTUM104 clone 2
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|AgeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|0.2 µl
 +
|BSA (100x)
 +
|-
 +
|13.8 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|4 µl
 +
|CHS- in pTUM104 clone 2
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|AgeI(20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|0.2 µl
 +
|BSA (100x)
 +
|-
 +
|13.3 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|5 µl
 +
|CHS- in pTUM104 clone 1
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|AgeI(20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|0.2 µl
 +
|BSA (100x)
 +
|-
 +
|12.3 µl
 +
|ddH2O
 +
|}
 +
 +
expected bands:
 +
*pTUM104: about 5800bp
 +
*4CL: 1685bp
 +
*CHS: 1173bp
 +
*OMT: 1222bp
 +
[[File: TUM_12_20120719_4CL,_CHS,_OMT_in_pYES_clones1.jpg]]
 +
[[File: TUM_12_20120719_4CL,_CHS,_OMT_in_pYES_clones2.jpg]]
 +
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Transformation of ligationsproducts of 4CL and PAL in pSB1C3-RFC25 and of PAL in pTUM104 in ''E.coli'' (see July, 18th)===
 +
Investigator: Daniela
 +
 +
*4CL+ (PCR15) in pSB1C3-RFC25 (P132) ->Chlp
 +
*4CL- (PCR16) in pSB1C3-RFC25 (P132) ->Chlp
 +
*PAL+ (PCR32) in pSB1C3-RFC25 (P133) ->Chlp
 +
*PAL- (PCR33) in pSB1C3-RFC25 (P133) ->Chlp
 +
*PAL+ (PCR32) in pTUM104(P72) ->Amp
 +
*PAL- (PCR33) in pTUM104(P72) ->Amp
 +
*negative control: P72 ->Amp
 +
*negative control: P132 ->Chlp
 +
*negative control: P133 ->Chlp
 +
 +
* adding 5µl ligation product in 100 µl competent XL blue E.coli cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 30 min for ampicillin and 45 min for chloramphenicol, 180 rpm
 +
* plate on agar with ampicillin or chloramphenicol over night
 +
 +
</div>
 +
 +
== '''Friday, July 20th''' ==
 +
<div class="constitutive_promoter">
 +
===Preparative Digestion of PGK1-P, TEF2-T, TEF1-P and gelelectrophoresis===
 +
 +
'''Investigator:''' Georg
 +
 +
* Reaction batch for digestion:
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|3,3 µl
 +
|PstI-HF (NEB)
 +
|-
 +
|3,3 µl 
 +
|XbaI (NEB)
 +
|-
 +
|13,2µl
 +
|NEB-4 buffer
 +
|-
 +
|1,32 µl
 +
|100 x BSA (NEB)
 +
|-
 +
|44,88 µl
 +
|dd H20
 +
|-
 +
|=72µl
 +
|'''TOTAL'''
 +
|}
 +
* 20 µl preparative mastermix were mixed with 20 µl of plasmid-DNA
 +
* Preparative gelelectrophoresis was processed for 90 min at 70 V
 +
 +
[[File:20120720 tef2 pgk1.PNG]]
 +
[[File:20120720 TEF1-Promoter.PNG]]
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Gel purification of Xba1/ Age1 digested P79  ===
 +
'''Investigator:''' Roman
 +
 +
'''Aim of the Experiment:''' Aim of the experiment is the purification of the double digested vector p79 (pSB1C3_RFC25_Linker), in which the N- terminal SS is to be cloned.
 +
 +
'''Operational sequence:'''
 +
* The mixture was seperated by gel electrophoresis (LMP agarose)
 +
* The DNA was cut out of the gel (2 pieces) and weight ==> 200 mg each piece.
 +
* Afterwards the plasmid DNA was extracted as described in the Quiagen Gel Purification protocol.Elution was performed in two steps (à 25 µl) with elution buffer (heated up to 40°). To improve the yeald of plasmid DNA during the elution, the column was incubated at RT for about 4 minutes before centrifugation
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Miniprep of transformed ''E.&nbsp;coli'' XL1-Blue with pTUM104 new from P50 tube (4x), BBa_K365005 (4x), BBa_K207000 (1x)and BBa_K207001 (1x)  ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the study:''': Miniprep
 +
 +
'''Operational sequence:'''
 +
 +
* Miniprep has been performed after manufacturer's protocol. QIAGEN - QIAprep Spin Miniprep Kit.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Analytical digestion and gelelectrophoresis of P152, P155, P156, P157, P158, P159, P160, P161  ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the study:''' Analytical digestion and gelelectrophoresis of P152, P155, P156, P157, P158, P159, P160, P161, to prove the insert and backbone size.
 +
 +
'''Operational sequence:'''
 +
 +
* Analytical digestion and gelelectrophoresis were performed after lab's standard protocol.
 +
 +
* Digestion with XbaI and PstI-HF in Buffer 4.
 +
 +
[[File:TUM12 20120720 anal.jpg|500px]]
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Picking from the overnight transformation of the ligated P126+PCR31  ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the study:''' Picking from the overnight transformation of the ligated P126+PCR31, to see whether ligation is successful or not.
 +
 +
'''Operational sequence:'''
 +
 +
* Picking and overnight culture after standard laboratory's protocol. (AmpR LB-medium)
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== PCR of P23 (LexA, BBa_K105005) to indroduce RFC25 pre- and suffix because last ligation was not successful ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the experiment:''' Last ligation with LexA was not successful and the tube with the PCR product was empty from the ligation, so one has to do another PCR for another ligation.
 +
 +
'''Operational sequence:'''
 +
 +
* Clone 3 (tube P23) of BBa_K105005 (LexA) has beed choosen for the PCR.
 +
 +
'''PCR reaction mixture'''
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|10&nbsp;µl
 +
|5x OneTaq Standard Reaction Buffer
 +
|-
 +
|1&nbsp;µl
 +
|10&nbsp;mM dNTPs
 +
|-
 +
|1&nbsp;µl
 +
|10&nbsp;µM Forward Primer
 +
|-
 +
|1&nbsp;µl
 +
|10&nbsp;µM Reverse Primer
 +
|-
 +
|0.25&nbsp;µL
 +
|OneTaq Hot Start DNA Polymerase (Finally: 1.25&nbsp;units/50&nbsp;µL)
 +
|-
 +
|1&nbsp;µl
 +
|Plasmid DNA (BBa_K105005) from P23 (Clone 3)
 +
|-
 +
|35.75&nbsp;µL
 +
|ELGA Water
 +
|-
 +
|=50&nbsp;µL
 +
|'''TOTAL'''
 +
|}
 +
 +
* The PCR program was performed after following scheme:
 +
{|cellspacing="0" border="1"
 +
|Initial denaturation
 +
|94&nbsp;°C
 +
|30&nbsp;s
 +
|-
 +
|30 cycles
 +
|94&nbsp;°C
 +
|30&nbsp;s
 +
|-
 +
|
 +
|55&nbsp;°C
 +
|60&nbsp;s
 +
|-
 +
|
 +
|68&nbsp;°C
 +
|60&nbsp;s
 +
|-
 +
|Final extension
 +
|68&nbsp;°C
 +
|5&nbsp;min
 +
|-
 +
|Hold
 +
|4&nbsp;°C
 +
|overnight
 +
|}
 +
 +
* Freezed at -20&nbsp;°C. Stil has to be purified with PCR purification kit on the next day
 +
</div>
 +
 +
<div class="vector_design">
 +
 +
===Control digest of pTUM104 (P153 and P154)===
 +
Investigator: Mary, Ingmar, Daniela
 +
 +
'''Aim of the experiment:''' Test whether the vector pTUM104 is right.
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.5 µl
 +
|P153
 +
|-
 +
|0.25 µl
 +
|AgeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|16.25 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.5 µl
 +
|P153
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|14.25 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.5 µl
 +
|P153
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|AgeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|14 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.5 µl
 +
|P153
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|14 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.5 µl
 +
|P153
 +
|-
 +
|0.25 µl
 +
|NgoMIV (10U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|16.25 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|1.5 µl
 +
|P153
 +
|-
 +
|0.25 µl
 +
|NgoMIV (10U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|0.25 µl
 +
|SpeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|13.75 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.8 µl
 +
|P154
 +
|-
 +
|0.25 µl
 +
|AgeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|14.95 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.8 µl
 +
|P154
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|12.95 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.8 µl
 +
|P154
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|AgeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|12.7 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.8 µl
 +
|P154
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|12.7 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.8 µl
 +
|P154
 +
|-
 +
|0.25 µl
 +
|NgoMIV (10U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|14.95 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.8 µl
 +
|P154
 +
|-
 +
|0.25 µl
 +
|NgoMIV (10U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|0.25 µl
 +
|SpeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|12.45 µl
 +
|ddH2O
 +
|}
 +
 +
*P153
 +
[[File:TUM12_20120720analyt.verdau_P153.jpg]]
 +
 +
*P154
 +
[[File:TUM12_20120720_ana_verdau_P154.jpg]]
 +
 +
The vector seems to be correct. However NgoMIV did not digest the plasmid properly.
 +
</div>
 +
 +
== '''Saturday, July 21st''' ==
 +
 +
<div class="light_switchable_promoter">
 +
=== Miniprep of transformed E. coli XL1-Blue with ligation product of P126+PCR31 ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the study:''' Miniprep
 +
 +
'''Operational sequence:'''
 +
 +
* Miniprep has been performed after manufacturer's protocol. QIAGEN - QIAprep Spin Miniprep Kit.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Analytical digestion and gelelectrophoresis of the minipreps of transformed E. coli XL1-Blue with ligation product of P126+PCR31 ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the experiment:''' To see whether ligation ist successful.
 +
 +
* Reaction batch for each plasmid:
 +
{|cellspacing="0" border="1"
 +
|'''Reagent'''
 +
|'''Volume in µl'''
 +
|-
 +
|Tango Buffer 10x
 +
|4&nbsp;µl
 +
|-
 +
|NdeI (Fermentas)
 +
|0.5&nbsp;µl
 +
|-
 +
|BamHI (Fermentas)
 +
|0.5&nbsp;µl
 +
|-
 +
|Plasmid DNA
 +
|2.5&nbsp;µl
 +
|-
 +
|ddH2O
 +
|12.5&nbsp;µl
 +
|-
 +
|'''TOTAL'''
 +
|'''20&nbsp;µl'''
 +
|}
 +
 +
* Incubation at 37&nbsp;°C for 1&nbsp;h 30&nbsp;min.
 +
 +
* Analytical gelelectrophoresis at 90&nbsp;V for 1&nbsp;h.
 +
 +
* Order of gel-pockets:
 +
{|cellspacing="0" border="1"
 +
|100&nbsp;bp ladder
 +
|P172
 +
|P173
 +
|P174
 +
|1000&nbsp;bp ladder
 +
|-
 +
|
 +
|'''corrupt'''
 +
|'''corrupt'''
 +
|'''corrupt'''
 +
|
 +
|}
 +
 +
[[File:TUM12_20120721_anal.jpg|500px]]
 +
 +
* Ligation was not successful!
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== PCR purification of PCR products from P23 ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the experiment''' Purification of PCR products from P23.
 +
 +
'''Operational sequence:'''
 +
 +
* The 4 tubes with PCR products of P23 were purificated with the PCR purification kit from Qiagen after manufacturer'S protocol.
 +
</div>
 +
 +
== '''Sunday, July 22nd''' ==
 +
 +
</div>
 +
<div class="coumaryl">
 +
=== Preparative digest of pTUM104 RFC25 and ligation with PCR products PCR 15 - 20 and 32+33 ===
 +
 +
'''Investigator: Ingmar'''
 +
 +
'''Aim of the experiment:''' Intsert the genes of PAL, 4CL, CHS and OMT in pTUM104 RFC 25 in order to test their expression in yeast.
 +
 +
'''Preparative digest of P 154 with XbaI and NgoMIV and of P 153 with XbaI and PstI'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|7 µl
 +
|ddH2O
 +
|-
 +
|2.5 µl
 +
|NEB 4 buffer
 +
|-
 +
|2.5 µl
 +
|Tango buffer
 +
|-
 +
|4 µl
 +
|XbaI (10 U/µl)
 +
|-
 +
|4 µl
 +
|NgoMIV (10 U/µl)
 +
|-
 +
|30 µl
 +
|P 153
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|10 µl
 +
|ddH2O
 +
|-
 +
|4 µl
 +
|Tango buffer
 +
|-
 +
|2 µl
 +
|XbaI (10 U/µl)
 +
|-
 +
|4 µl
 +
|PstI (10 U/µl)
 +
|-
 +
|20 µl
 +
|P 154
 +
|}
 +
 +
Incubation: 37 °C, 3 h
 +
 +
'''DNA preparative gel electrophoresis'''
 +
*gel: 1% with LMP-agarose
 +
*Each digest product was mixed with an adequate volume of DNA loading buffer and loaded into the gel
 +
*The separation process lasted 90 min at 70 V.
 +
 +
[[File:xxx.jpg|500px|Gel picture of control digest with PstI]]
 +
*All digest products show the expected bonds at 5849 bp (digest with XbaI and NgoMIV) and 5785 bp (digest with XbaI and PstI) respectively.
 +
 +
'''Gelextration'''
 +
*cut the bands
 +
*QIAquick Gel Extractrion Kit was used
 +
** step 6 was left out
 +
** step 9: 30µl buffer, 4 min incubation
 +
 +
Products were named as follows:
 +
* P154 digested with XbaI and NgoMIV: P175
 +
* P153 digested with XbaI and PstI: P176
 +
 +
'''Ligation'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''PCR product'''
 +
|'''Volume'''
 +
|'''Vector'''
 +
|'''Volume'''
 +
|'''Volume T4 DNA Ligase'''
 +
|'''Volume T4 DNA Ligase buffer'''
 +
|'''Volume ddH20'''
 +
|'''New Plasimd Number'''
 +
|-
 +
|PCR15
 +
|5.25
 +
|P 176
 +
|2.75
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 177
 +
|-
 +
|PCR16
 +
|5.25
 +
|P 176
 +
|2.75
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 178
 +
|-
 +
|PCR 17
 +
|7.2
 +
|P 175
 +
|0.8
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 179
 +
|-
 +
|PCR 18
 +
|7.2
 +
|P 175
 +
|0.8
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 180
 +
|-
 +
|PCR 19
 +
|7.1
 +
|P 175
 +
|0.9
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 181
 +
|-
 +
|PCR 20
 +
|7.1
 +
|P 175
 +
|0.9
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 182
 +
|-
 +
|PCR 32
 +
|7.25
 +
|P 175
 +
|0.75
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 183
 +
|-
 +
|PCR 33
 +
|7.5
 +
|P 175
 +
|0.5
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 184
 +
|-
 +
|}
 +
</div>
 +
</div>
 +
 +
=Week 7=
 +
<!--
 +
<p class="limonene">'''Limonene (7 Experiments):'''</p>
 +
*
 +
 +
<p class="coumaryl">'''Xanthohumol (18 Experiments):'''</p>
 +
*
 +
 +
<p class="constitutive_promoter">'''Constitutive Promoter (2 Experiments):'''</p>
 +
*
 +
 +
<p class="light_switchable_promoter">'''Light Switchable Promoter (15 Experiments):'''</p>
 +
*
 +
 +
<html><a class="WDetails" href="#Week_7" id="Week_7">Show Details ...</a></html>
 +
-->
 +
<div class="week" id="WWeek_7">
 +
== '''Monday, July 23rd''' ==
 +
 +
<div class="coumaryl">
 +
===Miniprep of PAL, 4CL in pSB1C3 and PAL in pTUM104===
 +
 +
Investigator: Mary
 +
 +
'''Aim of the experiment''': Extract plasmids (pTUM104 and pSB1C3-RFC25) that contain 4CL and PAL´
 +
 +
The day before one clone were picked for each enzyme from plates from 19th July 2012.
 +
 +
QIAprepS Spin Miniprep Kit
 +
*step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)
 +
 +
Concentration:
 +
*4CL+ (PCR15) in pSB1C3-RF25 (P132): c = 340 ng/µl -> new name of Ligationproduct after Miniprep: P185
 +
*4CL- (PCR16) in pSB1C3-RF25 (P132): c = 385 ng/µl -> new name of Ligationproduct after Miniprep: P186
 +
*PAL+ (PCR32) in pSB1C3-RF25 (P133): c = 395 ng/µl -> new name of Ligationproduct after Miniprep: P187
 +
*PAL- (PCR33) in pSB1C3-RF25 (P133): c = 255 ng/µl -> new name of Ligationproduct after Miniprep: P188
 +
 +
*PAL+ (PCR32) in pTUM104 (P72): c = 190 ng/µl
 +
*PAL- (PCR33) in pTUM104 (P72): c = 238 ng/µl
 +
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Control digest of 4CL, PAL in pSB1C3-RFC25 and PAL in pTUM104===
 +
 +
Investigator: Mary
 +
 +
'''Aim of the experiment:''' Test whether the ligation of 4CL, PAL in pSB1C3-RFC25 and PAL in pTUM104 was successful
 +
 +
Plasmids are taken from miniprep from 23.07.2012
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|4CL+ in pSB1C3-RFC25 / 4CL- in pSB1C3-RFC25
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|13 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|PAL+ in pSB1C3-RFC25 / PAL- in pSB1C3-RFC25
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|AgeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|13 µl
 +
|ddH2O
 +
|}
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|PAL+ in pSB1C3-RFC25 / PAL- in pSB1C3-RFC25
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|AgeI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|13 µl
 +
|ddH2O
 +
|}
 +
 +
expected bands:
 +
*pTUM104: about 5800bp
 +
*pSB1C3-RFC25: 2070bp
 +
*4CL: 1685bp
 +
*PAL: 2151bp
 +
 +
[[File:TUM12_120723_kontrollverdau_wdh.jpg|500px]]
 +
 +
Ligation of PAL+/- in pTUM104 was not succesful
 +
Ligation of PAL+/- and 4CL+/- in pSB1C3-RFC25 were succesful! (pSB1C3 is overlapping with PAL)
 +
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===APT Solubilisation and Transformation===
 +
 +
Investigator: Mary
 +
 +
'''Aim of the experiment:''' solubilisation of the synthetic gene of APT and transformation of sent plasmid (including APT) in ''E.coli'' to copy it if necessary
 +
 +
short zentrifugation of sent product and adding 50µl bidest. H2O to it (5µg in 50µl = concentration of 0.1 µg/µl)
 +
the dissolved product was named as G1 (as Geneproduct number 1) and stored at -20°C
 +
 +
2µl of solved product used for transformation in Ecoli (Kit of Quiagen) and Amp-resistance (said GeneArt)
 +
Plating cells on Agar with Amp, 37°C over night
 +
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Preparative digest of APT===
 +
 +
Investigator: Mary
 +
 +
'''Aim of the experiment:''' Extraction of the sequence of APT out of the sent plasmid.
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
| 10µl
 +
|ddH2O
 +
|-
 +
|4 µl
 +
|NEB 4 buffer
 +
|-
 +
|4 µl
 +
|BSA (10x)
 +
|-
 +
|1 µl
 +
|XbaI (20 U/µl)
 +
|-
 +
|1 µl
 +
|AgeI (20 U/µl)
 +
|-
 +
|20 µl
 +
|solubilised APT-product (see Solubilisation APT)
 +
|}
 +
 +
Incubation: 37°C, 2.5h
 +
 +
Bond at 1244bp as expected:
 +
 +
[[File:TUM12_120723_prepGel_von_APT.jpg|500px]]
 +
 +
The bond was cutted out of the gel and stored at -20°C (P189)
 +
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Transformation of  ligationsproducts of 4CL, CHS, OMT  and PAL in pTUM104 in ''E.Coli'' (Ligation see 22th of July)===
 +
 +
Investigator: Mary
 +
 +
'''Aim of the experiment:''' ligation of enzymes in pTUM104 to transform it into yeast if this was successful.
 +
* 4CL+ (PCR15) in pYES (P176) -> new name: P177
 +
* 4CL- (PCR16) in pYES (P176) -> new name: P178
 +
* CHS+ (PCR17) in pYES (P175) -> new name: P179
 +
* CHS- (PCR18) in pYES (P175) -> new name: P180
 +
* OMT+ (PCR19) in pYES (P175) -> new name: P181
 +
* OMT- (PCR20) in pYES (P175) -> new name: P182
 +
* PAL+ (PCR32) in pYES (P175) -> new name: P183
 +
* PAL- (PCR33) in pYES (P175) -> new name: P184
 +
 +
* adding 5µl ligation product in 100 µl competent XL blue ''E.coli'' cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 30 min 180 rpm
 +
* plate on agar with ampicillin and incubate over night at 37°C
 +
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
===Gel- purification of hybridized oligos: SV40 SS (Tube PCR34)===
 +
'''Investigator: Roman'''
 +
'''Aim of the experiment:''' Purification of the hybridized oligos in order to make them ready for ligation in pSB1C3
 +
'''Operational Sequence:'''
 +
* oligos were seperated by gel electrophoresis (LMP agarose)
 +
* picture:
 +
[[File:TUM12_20120723_PCR34.png|200px|preparative gel electrophoresis]]
 +
* afterwards the DNA was cut out and extracted with the Quiagen Gel- purification kit as described in the manufacturer's protocol. Elution was performed in two steps á 25 µl elution buffer. DNA was collected in one tube, which was annotated with PCR34 purif.
 +
* determined concentration (NanoDrop): ca. 385 ng/µl
 +
</div>
 +
 +
== '''Tuesday, July 24nd''' ==
 +
<div class="constitutive_promoter">
 +
=== Gelextraction of preparative digested ADH1-P, CYC1-T, TEF2-P, TEF1-P,PGK1-P===
 +
 +
'''Investigator:''' Georg
 +
*Plasmid-DNA was extracted according to Quiaquick gel extraction kit
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
===Nanodrop: measuring concentration of P128-P131, P168-P169===
 +
 +
''' Investigator:''' Georg
 +
*P128: 28,3 ng/µl
 +
*P129: 21  ng/µl
 +
*P130: 4,2 ng/µl
 +
*P131: 4,7 ng/µl
 +
*P168: 3,5 ng/µl
 +
*P169: 4,2 ng/µl
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Gelextraction of APT digested with Xbal and AgeI (P189)===
 +
Investigator: Daniela
 +
 +
'''Gelextration'''
 +
*QIAquick Gel Extractrion Kit was used
 +
*the product was named P189
 +
*concentration: c = 9.1 ng/µl
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Ligation of APT (P189) in pSB1C3-RFC25 (P133)===
 +
Investigator: Daniela
 +
 +
APT (P189) in pSB1C3-RFC25 (P133)
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|0.86 µl
 +
|P133
 +
|-
 +
|7.14 µl
 +
|APT (P189)
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|2 µl
 +
|T4-ligase buffer (10x)
 +
|-
 +
|9 µl
 +
|ddH2O
 +
|}
 +
Negative control:
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|0.86 µl
 +
|P133
 +
|-
 +
|16.14 µl
 +
|ddH2O
 +
|-
 +
|1 µl
 +
|T4 DNA Ligase
 +
|-
 +
|2 µl
 +
|T4-ligase buffer (10x)
 +
|}
 +
*water bath 16 °C
 +
</div>
 +
<div class="light_switchable_promoter">
 +
===NanoDrop determination of PCR34- PCR38===
 +
'''Investigator:'''Roman
 +
 +
'''Aim:''' Determination of the concentration of the samples previously to the ligation
 +
 +
Determined values:
 +
{|cellspacing="0" border="1"
 +
|'''PCR Product'''
 +
|'''Concentration in ng/µl'''
 +
|-
 +
|PCR34
 +
|385 (hybridized oligos)
 +
|-
 +
|PCR35
 +
|41,2
 +
|-
 +
|PCR36
 +
|39,5
 +
|-
 +
|PCR37
 +
|41,9
 +
|-
 +
|PCR38
 +
|57,4
 +
|}
 +
 +
* PCR38 will be used in following restriction digest
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
===Restriction digest of p123 and PCR38 with NgoMIV and Pst1===
 +
'''Investigator:''' Roman
 +
'''Aim of the experiment:''' To prepare samples of p123 (pSB1C3 RFC25) and PCR38 for a following ligation
 +
'''Operational sequence:'''
 +
p123 was digested in a 40 µl preparation (miniprep product):
 +
{|cellspacing="0" border="1"
 +
|'''Substance'''
 +
|'''Volume'''
 +
|-
 +
|Plasmid- DNA
 +
|20 µl
 +
|-
 +
|Enzyme NgoMIV
 +
|2 µl
 +
|-
 +
|Enzyme Pst1
 +
|2 µl
 +
|-
 +
|Buffer 4
 +
|4 µl
 +
|-
 +
|ddH2O
 +
|12 µl
 +
|}
 +
PCR38 was digested in a 50 µl preparation (PCR product)
 +
{|cellspacing="0" border="1"
 +
|'''Substance'''
 +
|'''Volume'''
 +
|-
 +
|Purified PCR product
 +
|25
 +
|-
 +
|Enzyme NgoMIV
 +
|2 µl
 +
|-
 +
|Enzyme Pst1
 +
|2 µl
 +
|-
 +
|Buffer 4
 +
|5 µl
 +
|-
 +
|ddH2O
 +
|16 µl
 +
|}
 +
 +
The preparations were incubated at 37°C for 3h and then stored at -20°C in box  The tubes were annotated with "p123_doubledigest_NgoMIV+Pst1_unpurified_20120724" and "PCR38_doubledigest_NgoMIV+Pst1_unpurified_20120724"
 +
Afterwards, the samples were load on a 1% universal agarose gel and separated at 100 V for ca. 45 min. Corresponding gel- bands were cut out and stored at -20°C over night until extraction.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
===Ligation of p151 and PCR34===
 +
'''Investigator:''' Roman
 +
 +
'''Aim:''' Ligation of fragments previously to a transformation
 +
 +
'''Operational Sequence:'''
 +
Length of fragments:
 +
* p151: 2070 bp; c = 9,5 ng/µl
 +
* PCR34: 67 bp; c = 385 ng/µl
 +
20 µl preparation:
 +
{|cellspacing="0" border="1"
 +
|'''Substance'''
 +
|'''Volume'''
 +
|-
 +
|p151- DNA
 +
|11 µl
 +
|-
 +
|PCR34- DNA
 +
|3 µl (out of a 1/100 dilution)
 +
|-
 +
|Ligase
 +
|0,5
 +
|-
 +
|T4 Ligase Buffer
 +
|2 µl
 +
|-
 +
|ddH2O
 +
|3,5 µl
 +
|}
 +
 +
Negative controls were prepared the same way, using 3 µl of ddH2O instead of PCR34- DNA.
 +
The preparation was incubated 30 min at room temperature and then over night at 16°C (water bath). Afterwards, the samples were stored at 4°C until the transformation.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
===Ligation of p126 and PCR31===
 +
'''Investigator:''' Roman
 +
 +
'''Aim:'''Ligation of fragments previously to a transformation
 +
 +
'''Operational Sequence:'''
 +
Length of fragments:
 +
* p126: 7961 bp; c = 46 ng/µl
 +
* PCR34: 306 bp; c = 11,3 ng/µl
 +
 +
10 µl preparation:
 +
{|cellspacing="0" border="1"
 +
|'''Substance'''
 +
|'''Volume'''
 +
|-
 +
|p126- DNA
 +
|2 µl
 +
|-
 +
|PCR31- DNA
 +
|1 µl
 +
|-
 +
|Ligase
 +
|0,5
 +
|-
 +
|T4 Ligase Buffer
 +
|1 µl
 +
|-
 +
|ddH2O
 +
|5,5 µl
 +
|}
 +
 +
Negative controls were prepared the same way, using 1 µl of ddH2O instead of PCR31- DNA.
 +
The preparation was incubated 30 min at room temperature and then over night at 16°C (water bath).
 +
</div>
 +
 +
<div class="limonene">
 +
===Ligation of p175 with "PCR1, 19.7.2012" and p144===
 +
'''Investigator:''' Roman
 +
 +
'''Aim:''' Ligation of fragments previously to a transformation
 +
 +
'''Operational Sequence:'''
 +
Length of fragments:
 +
* p175: 5900 bp; c = 107,3 ng/µl
 +
* PCR1: 1680 bp; c = 18 ng/µl
 +
* p144: ??? bp; c = ???
 +
 +
10 µl preparation:
 +
{|cellspacing="0" border="1"
 +
|'''Substance'''
 +
|'''Volume'''
 +
|-
 +
|p175- DNA
 +
|1 µl
 +
|-
 +
|PCR1/ p144- DNA
 +
|3 µl
 +
|-
 +
|Ligase
 +
|0,5
 +
|-
 +
|T4 Ligase Buffer
 +
|1 µl
 +
|-
 +
|ddH2O
 +
|4,5 µl
 +
|}
 +
'''Note:''' This preparation was not made optimal, due to use of a wrong fragment lengths (PCR1 and p144, respectively) during the calculation of the volumes.
 +
Negative controls were prepared the same way, using 3 µl of ddH2O instead of PCR fragment.
 +
The preparation was incubated 30 min at room temperature and then over night at 16°C (water bath).
 +
</div>
 +
<div class="coumaryl">
 +
===Preparation of yeast SCU Minimal Medium for Plates===
 +
Investigator: Katrin, Daniela
 +
 +
*The recipe was taken from the pYES2 manual.
 +
*The ingredients were dissolved in 900 ml dest. water (ELGA) corresponding to the recipe. Lysine was only available as lysine-dihydrochloride, therefore 0.149 g instead of 0.1 g were used. Uracil was omited.
 +
*The medium was divided in 2 x 450 ml. One will later be used as the induction medium through the addition of galactose the other one will be used as the non-induction medium (addition of glucose). Sugar solutions will be added after autoclaving to prevent maillard-reaction.
 +
*10 g agar and a magnetic stir bar were added to each preparation
 +
*Autoclaving.
 +
*Glucose solution: 10 g glucose were dissolved in 50 ml ELGA.
 +
*Galactose solution: 10 g galactose were dissolved in 50 ml ELGA.
 +
*No raffinose will be used.
 +
*Autoclaving
 +
</div>
 +
<div class="coumaryl">
 +
===Transformation of APT in pSB1C3-RFC25 (P190)===
 +
Investigator: Daniela
 +
 +
* APT (P189) in pSB1C3 (P133) -> new name: P190
 +
* Negative control P133
 +
 +
* adding 5µl ligation product in 100 µl competent XL blue ''E.coli'' cells
 +
* incubation 30 min on ice
 +
* 5 min at 37°C
 +
* adding cells in 1 ml LB (without antibiotica) and incubate at 37°C, 45 min 180 rpm
 +
* plate on agar with chloramphenicol and incubate over night at 37°C
 +
</div>
 +
<div class="coumaryl">
 +
 +
=== Picking Clones of 4CL, CHS, OMT and PAL in pTUM104 and APT in original plasmid===
 +
 +
'''investigator: ''' Daniela
 +
 +
'''Aim of the experiment:''' preculture over night for the miniprep next day
 +
 +
</div>
 +
 +
== '''Wednesday, July 25th''' ==
 +
<div class="light_switchable_promoter">
 +
=== Restriction digest of p123 with Xba1 and Age1 ===
 +
'''Investigator:''' Roman
 +
 +
'''Aim of the experiment:'''
 +
Aim of the experiment is the preparation of the vector pSB1C3 RFC25 (p123) for a ligation with the PCR fragment PCR34 (as repetition for the ligation of p151 with PCR34, which has probably not worked, due to low vector concentration.
 +
 +
'''Operational sequence:'''
 +
30 µl preparation for restriction digest of p123
 +
{|cellspacing="0" border="1"
 +
|'''Substance'''
 +
|'''Volume'''
 +
|-
 +
|p123- DNA
 +
|10 µl (results in ca. 5µg DNA)
 +
|-
 +
|Xba1- RE
 +
|1 µl
 +
|-
 +
|Age1- RE
 +
|1 µl
 +
|-
 +
|NEBuffer 4
 +
|3 µl
 +
|-
 +
|ddH2O
 +
|15 µl
 +
|}
 +
 +
The preparation was incubated at 37°C for 3,5 h. Afterwards the fragments were purificated by means of a preparative gel electrophoresis (see below).
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Restriction digest of p123 with Age1 and Pst1 ===
 +
'''Investigator:''' Roman
 +
 +
'''Aim of the experiment:'''
 +
Aim of the experiment is the preparation of the pSB1C3- Vector (p123) for a ligation with PCR38 (which has been digested with NgoMIV and Pst1).
 +
 +
'''Operational sequence:'''
 +
30 µl preparation for restriction digest of p123
 +
{|cellspacing="0" border="1"
 +
|'''Substance'''
 +
|'''Volume'''
 +
|-
 +
|p123- DNA
 +
|10 µl (results in ca. 5µg DNA)
 +
|-
 +
|Pst1- RE
 +
|1 µl
 +
|-
 +
|Age1- RE
 +
|1 µl
 +
|-
 +
|NEBuffer 4
 +
|3 µl
 +
|-
 +
|ddH2O
 +
|15 µl
 +
|}
 +
 +
The preparation was incubated at 37°C for 3,5 h. Afterwards the fragments were purificated by means of a preparative gel electrophoresis (see below).
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Gel extraction of PCR38 and p123 (both digested with NgoMIV and Pst1) ===
 +
'''Investigator:''' Roman
 +
 +
'''Aim of the experiment:'''
 +
The separated PCR38 and p123 (pSB1C3 RFC25) DNA- fragments from yesterday's restriction digest (both NgoMIV and Pst1) are to be extracted from agarose- gel (which has been stored at -20°C over night), to make them ready for further usage.
 +
 +
'''Operational sequence:'''
 +
The extraction was performed as described in the manual of the Quiagen Gel- extraction kit. Elution was performed with 30 µl of elution- buffer EB in two steps (à 15 µl), temperated at 50°C. Concentration was determined with NanoDrop:
 +
* PCR38: 17,3 ng/µl
 +
* p123:  37,2 ng/µl
 +
 +
Tubes were annotated with p191 (p123 NgoMIV/Pst1) and p192 (PCR38 NgoMIV/Pst1).
 +
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Gel purification of p123 (Xba1/Age1 double digest) and p123 (Age1/Pst1 double digest) ===
 +
'''Investigator:''' Roman
 +
 +
'''Aim of the experiment:'''
 +
Aim of the experiment is the purification of two different double digested p123 samples, to make them ready for ligation with the inserts PCR38 and PCR34, respectively.
 +
 +
'''Operational sequence:'''
 +
The samples were loaded on a 1% universal agarose gel and separated at 100 V for about 45 min. Afterwards, the corresponding gel- bands were cut out and weight. Extraction from gel was performed as described in the manufacturers protocoll (Quiagen gel extraction kit). Changes: Elution was performed in one step with 30µl ddH2O (autoclaved) temperated at 50°C.
 +
Concentrations were determined with NanoDrop:
 +
* p123 (Xba1/Age1): 70,8 ng/µl
 +
* p123 (Pst1/Age1): 59,1 ng/µl
 +
The tubes were annotated with p206 (p123 Age1/Pst1) and p207 (p123 Age1/Xba1).
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
=== Ligation of p123 (Age1/Pst1) and PCR38 ===
 +
'''Investigator:''' Roman
 +
 +
'''Aim of the experiment:''' Ligation of the fragments p123 and PCR38
 +
 +
'''Operational sequence:'''
 +
Length of fragments:
 +
* p123: 2070 bp; c = 59,1 ng/µl
 +
* PCR38: 617 bp; c = 17,3 ng/µl
 +
20 µl preparation:
 +
{|cellspacing="0" border="1"
 +
|'''Substance'''
 +
|'''Volume'''
 +
|-
 +
|p123- DNA
 +
|3 µl (ca. 180 ng vector- dna)
 +
|-
 +
|PCR38- DNA
 +
|10 µl
 +
|-
 +
|Ligase
 +
|0,5
 +
|-
 +
|T4 Ligase Buffer
 +
|2 µl
 +
|-
 +
|ddH2O
 +
|4,5 µl
 +
|}
 +
 +
Negative controls were prepared the same way, using 10 µl of ddH2O instead of PCR38- DNA.
 +
The preparation was incubated 30 min at room temperature and then over night at 16°C (water bath). Afterwards, the samples were stored at 4°C in the fridge until transformation.
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Ligation of p123 (Age1/Xba1) and PCR34 ===
 +
'''Investigator:''' Roman
 +
 +
'''Aim of the experiment:'''
 +
Ligation of fragments p123 and PCR34
 +
'''Operational sequence:'''
 +
Length of fragments:
 +
* p123: 2070 bp; c = 70,8 ng/µl
 +
* PCR34: 67 bp; c = 3,85 ng/µl (1:100 dilution of original sample)
 +
20 µl preparation:
 +
{|cellspacing="0" border="1"
 +
|'''Substance'''
 +
|'''Volume'''
 +
|-
 +
|p123- DNA
 +
|3 µl (ca. 210 ng vector- dna)
 +
|-
 +
|PCR34- DNA
 +
|6 µl (out of 1:100 dilution)
 +
|-
 +
|Ligase
 +
|0,5
 +
|-
 +
|T4 Ligase Buffer
 +
|2 µl
 +
|-
 +
|ddH2O
 +
|8,5 µl
 +
|}
 +
 +
Negative controls were prepared the same way, using 6 µl of ddH2O instead of PCR34- DNA.
 +
The preparation was incubated 30 min at room temperature and then over night at 16°C (water bath). Afterwards, the samples were stored at 4°C in the fridge until transformation.
 +
</div>
 +
 +
<div class="coumaryl">
 +
===Miniprep of 4CL, CHS, OMT, PAL in pTUM104 and APT in original plasmid from GeneArt===
 +
 +
Investigator: Katrin
 +
 +
'''Aim of the experiment''': extraction of plasmids (pTUM104) that contain 4CL, CHS, OMT, PAL; extraction of original plasmid from GeneArt with APT
 +
 +
QIAprepS Spin Miniprep Kit
 +
*step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)
 +
the Minipreps were named as follows:
 +
*P193: 4CHL+ in pSB1C3-RFC25 (c = 110,7 ng/µl)
 +
*P194: 4CL- in pYEs (c = 342,8 ng/µl)
 +
*P195: CHS+ in pYEs (c = 423,6 ng/µl)
 +
*P196: CHS- in pYEs (c = 92,1 ng/µl)
 +
*P197: OMT+ in pYEs (c = 394,6 ng/µl)
 +
*P198: OMT- in pYEs (c = 183,0 ng/µl)
 +
*P199: PAL+ in pYEs (c = 138,4 ng/µl)
 +
*P200: PAL- in pYEs (c = 464,5 ng/µl)
 +
*P201: APT in original plasmid (c = 260,0 ng/µl)
 +
 +
</div>
 +
 +
<div class="limonene">
 +
===Transformation of Ligation-products into ''E.coli'' XL-1 Blue===
 +
 +
'''Investigator: Andrea'''
 +
 +
* for each Biobrick 100 µl cells were used and pooled together with 5 µl of ligation product from the 24th of july
 +
(3 hours at 16°C and over night at 4 °C) and from the 19th of july (5 days at 16°C)
 +
 +
* Incubation on ice for 30 min
 +
 +
* 5 min heat shock at 37 °C
 +
 +
* cells were prefilled with 1 ml of LB-medium and incubated in a cell-culture shaker at 37 °C for 40 min
 +
 +
* 100 µl of these cell suspension were plated on antibiotic selection plates (ligations in pTUM104: Ampicillin; ligations in pSB1C3: Chloramphenicol)
 +
 +
* cell suspension was centrifuged at 13000 rpm for 90 s for resuspending the pellet with 100 µl LB and plating also
 +
 +
* incubation at 37 °C overnight
 +
</div>
 +
 +
== '''Thursday, July 26th''' ==
 +
 +
<div class="light_switchable_promoter">
 +
=== Transformation of ligation products of P126+PCR31, P151+PCR34, P123+PCR38 and P123+PCR34 in ''E.&nbsp;coli'' XL1-Blue ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the experiment:'''Transformation of ligation products of P126+PCR31 (AmpR), P151+PCR34 (CamR), P123+PCR38 (CamR) and P123+PCR34 (CamR) in ''E.&nbsp;coli'' XL1-Blue
 +
 +
'''Operational sequence:'''
 +
 +
* Performed after lab's standard protocol.
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
=== Miniprep of APT in pSB1C3-RFC25 and control digestion with Xba1 and Pst1===
 +
 +
Investigator: Mary
 +
 +
'''Aim of the experiment''': extraction of plasmid (pSB1C3-RFC25) that contains APT and testing if ligation was succesful;
 +
three clones were picked the day before
 +
 +
'''Miniprep'''
 +
 +
QIAprepS Spin Miniprep Kit
 +
*step 3: invert 2-3 times (don't shake to avoid destruction of genomic DNA)
 +
the Minipreps were named as follows:
 +
*APT in pSB1C3-RFC25 clone 1: c=96 ng/µl
 +
*APT in pSB1C3-RFC25 clone 2: c=108 ng/µl
 +
*APT in pSB1C3-RFC25 clone 3: c=97 ng/µl
 +
 +
'''Control digest'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|4CL+/-, PAL+/-, CHS+/-, OMT+/-, APT+/-
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|13 µl
 +
|ddH2O
 +
|}
 +
 +
expected bands:
 +
*pSB1C3-RF25: 2070bp
 +
*APT: 1244bp
 +
 +
[[File:TUM12_120726_APT_in_pSB1C3.jpg‎|500px]]
 +
</div>
 +
 +
<div class="coumaryl">
 +
===Control digest of 4CL, PAL, CHS, OMT, APT in pTUM104===
 +
 +
Investigator: Mary
 +
 +
'''Aim of the experiment:''' Test whether the ligation of 4CL, PAL, CHS, OMT, APT in pTUM104 was successful
 +
 +
Plasmids are taken from miniprep from 25.07.2012
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|4CL+/-, PAL+/-, CHS+/-, OMT+/-, APT+/-
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|13 µl
 +
|ddH2O
 +
|}
 +
 +
expected bands:
 +
*pYES: about 5800bp
 +
*4CL: 1685bp
 +
*PAL: 2151bp
 +
*CHS: 1173bp
 +
*OMT: 1222bp
 +
*APT: 1244bp
 +
 +
[[File:TUM12_120726_pYDS_controldigest.jpg|500px]]
 +
</div>
 +
 +
<div class="coumaryl">
 +
=== Pour on yeast agarplates for selection of yeast cells ===
 +
 +
Investigator: Mary
 +
 +
'''Aim of the experiment:''' prepare selection-agarplates without Uracil (Some plates including glucose, some including galactose)
 +
 +
it was strange that the agar was still pretty liquid after waiting for one hour.
 +
 +
see pYES-manual
 +
</div>
 +
 +
<div class="limonene">
 +
 +
===Picking of clones of transformation from 25.07.===
 +
 +
'''Investigator:''' Lara
 +
 +
'''Aim:''' Amplification of clones for extraction of plasmids and subsequent proof of functional ligation.
 +
 +
6 clones were picked for each ligation (PCR1 in pYESnew, PCR2 (p144) in pYESnew and PCR in pSB1C3. Clones containing pYESnew were put into 4 ml LB with 4 µl of ampillicin stock solution, clones containing pSB1C3 were put into chloramphenicol-containing media. Incubation at 37 °C over night.
 +
</div>
 +
 +
<div class="limonene">
 +
===Transformation of plasmids P40&P42 into ''E.coli'' XL1 blue===
 +
 +
'''Investigator:''' Lara
 +
 +
'''Aim:'''
 +
 +
Transformation of plasmids P40 and P42 into ''E.coli'' XL1 blue to get plasmids of higher quality. P40&P42 are directly extracted from the expression strain BL21(DE). The quality of the plasmids might not be sufficient for Quickchange site-directed mutagenesis. Therefore transformation into ''E.coli'' XL1 blue and subsequent plasmid preparation to get plasmids with sufficient quality for SDM.
 +
 +
'''Procedure:'''
 +
 +
100 μl of competent XL1 blue cells were thawed on ice. 1 µl plasmid DNA (P40 or P42) was added. Incubation for 30 min on ice. 5 min heatshock at 37°C. 1 ml of LB medium without antibiotic was added, incubation for 45 min at 180 rpm/37°C.
 +
After incubation, 100 µl of the cell suspension were plated on antibiotic containing plates (P40: Kan, P42: Amp). The remaining solution was centrifuged for 60 sec, resuspended in 100 µl LB and plated, as well.
 +
Incubation at 37 °C over night.
 +
</div>
 +
 +
== '''Friday, July 27th''' ==
 +
 +
<div class="light_switchable_promoter">
 +
=== Picking from the overnight transformation of ligated P126+PCR31, P151+PCR34, P123+PCR38 and P123+PCR34 in ''E.&nbsp;coli'' XL1-Blue ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the experiment''' Picking of transformated ''E.&nbsp;coli''XL1-Blue with ligation product of P126+PCR31 (AmpR), P151+PCR34 (CamR), P123+PCR38 (CamR) and P123+PCR34 (CamR).
 +
 +
'''Procedure'''
 +
 +
* Colonies were picked with pipette tips and transformed into a new cell-culture tube with 4&nbsp;ml of LB-medium and antibiotics and were put overnight in a 180&nbsp;rpm cell culture shaker at 37&nbsp;°C. P126+PCR31 (AmpR), P151+PCR34 (CamR), P123+PCR38 (CamR) and P123+PCR34 (CamR)
 +
</div>
 +
 +
<div class="light_switchable_promoter">
 +
 +
=== Transferring ''E.&nbsp;coli'' XL1-Blue colonies, transformed with BBa_I15008 (heme oxygenase) and BBa_K105005 (LexA), from old antibiotic plates on new plates ===
 +
 +
'''Investigator:''' Jeff
 +
 +
'''Aim of the experiment''' With a sterile incolation loop the colonies were transferred on a new antibiotic plate, because the plates are already 4&nbsp;weeks old.
 +
 +
'''Procedure'''
 +
 +
* Incolation loop was put for few second into the flame of a Bunsen burner
 +
 +
* Colonies from plate with BBa_I15008 (heme oxygenase, KanR) and BBa_K105005 (LexA, AmpR) were transferred on a new antibiotic plate
 +
 +
* Overnight-culture at 37&nbsp;°C
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
===Miniprep and control digest of PAL in pYES picked on thursday 26th July===
 +
 +
Investigator: Ingmar
 +
 +
'''Aim of the experiment:''' Test whether the ligation of PAL in pTUM104 was successful
 +
 +
'''Miniprep'''
 +
Using a Quiagen Miniprep kit a plasmid isolation of two picked clones of the PAL+ in pYes2 RFC25 ligation was done. The miniprep product of the first clone was named P219, the one of the second clone P220. Both were used for the following control digest.
 +
 +
'''control digest'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5 µl
 +
|PAL+
 +
|-
 +
|0.25 µl
 +
|Xbal (20U/µl)
 +
|-
 +
|0.25 µl
 +
|PstI (20U/µl)
 +
|-
 +
|2 µl
 +
|NEB4 (10x)
 +
|-
 +
|2 µl
 +
|BSA (10x)
 +
|-
 +
|13 µl
 +
|ddH2O
 +
|}
 +
 +
expected bands:
 +
*pYES:5819bp
 +
*PAL: 2151bp
 +
 +
[[File:TUM12_Xanthohumol_P219_und_P220__27.07.2012.jpg|500px]]
 +
 +
As the picture only shows one band at the expected length of the vecotr backbone the ligation was not successfull and will be repeated with a reduced quotient of insert to vector of 3.
 +
</div>
 +
 +
<div class="coumaryl">
 +
 +
=== Ligation of PAL+ (PCR32) and APT (P189) with pYes2 RFC25 (P175) ===
 +
 +
'''Investigator: Ingmar'''
 +
 +
'''Aim of the experiment:''' Insert the genes of PAL and APT in pYEs2 RFC 25 in order to test their expression in yeast.
 +
 +
'''Ligation'''
 +
 +
{|cellspacing="0" border="1"
 +
|'''PCR product'''
 +
|'''Volume'''
 +
|'''Vector'''
 +
|'''Volume'''
 +
|'''Volume T4 DNA Ligase'''
 +
|'''Volume T4 DNA Ligase buffer'''
 +
|'''Volume ddH20'''
 +
|'''New Plasimd Number'''
 +
|-
 +
|PAL+ (PCR32)
 +
|6.6
 +
|P 175
 +
|1.4
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 221
 +
|-
 +
|APT (P 189)
 +
|7.06
 +
|P 175
 +
|0.94
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|P 222
 +
|-
 +
|control: ddH2O
 +
|7
 +
|P 175
 +
|1
 +
|1 µl
 +
|2 µl
 +
|9 µl
 +
|
 +
|-
 +
|}
 +
The Ligation product of PAL+ and pYes was labeled P233, the one of APT and pYes P234.
 +
</div>
 +
 +
<div class="limonene">
 +
 +
=== Plasmid extraction of plasmids PCR1/pTUM104, P144/pTUM104, PCR1/pSB1C3 ===
 +
 +
'''Investigator: Lara'''
 +
 +
'''Aim of the experiment:''' Extract plasmids from ligation of PCR1 in pYES, P144 in pTUM104 and PCR1 in pSB1C3 for further analytical restriction digest. (6 clones were picked for each ligation and plasmids subsequently extracted.)
 +
 +
'''Prodedure:''' Plasmids were extraced by using Qiagen plasmid miniprep kit.
 +
 +
Following concentrations were obtained:
 +
 +
1_1 - 1_6
 +
*PCR1 in pYESnew clone 1: c=66 ng/µl
 +
*PCR1 in pYESnew clone 2: c=182 ng/µl
 +
*PCR1 in pYESnew clone 3: c=98 ng/µl
 +
*PCR1 in pYESnew clone 4: c=326 ng/µl
 +
*PCR1 in pYESnew clone 5: c=87 ng/µl
 +
*PCR1 in pYESnew clone 6: c=156 ng/µl
 +
 +
2_1 - 2_6
 +
 +
*P144(PCR2) in pYESnew clone 1: c=195 ng/µl
 +
*P144(PCR2) in pYESnew clone 2: c=202 ng/µl
 +
*P144(PCR2) in pYESnew clone 3: c=198 ng/µl
 +
*P144(PCR2) in pYESnew clone 4: c=77 ng/µl
 +
*P144(PCR2) in pYESnew clone 5: c=95 ng/µl
 +
*P144(PCR2) in pYESnew clone 6: c=200 ng/µl
 +
 +
3_1 -3_6
 +
*PCR1 in pSB1C3-RFC25 clone 1: c=116 ng/µl
 +
*PCR1 in pSB1C3-RFC25 clone 2: c=110 ng/µl
 +
*PCR1 in pSB1C3-RFC25 clone 3: c=116 ng/µl
 +
*PCR1 in pSB1C3-RFC25 clone 4: c=112 ng/µl
 +
*PCR1 in pSB1C3-RFC25 clone 5: c=139 ng/µl
 +
*PCR1 in pSB1C3-RFC25 clone 6: c=80 ng/µl
 +
 +
</div>
 +
<div class="limonene">
 +
 +
=== Restriction digest of plasmids from 6 clones each of PCR1/pTUM104new, P144(PCR2)/pTUM104new, PCR1/pSB1C3 ===
 +
 +
'''Investigator:''' Lara
 +
 +
'''Aim of the experiment:''' Analytical restriction digest of plasmids to check for insert.
 +
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|3 µl
 +
|DNA
 +
|-
 +
|2 µl
 +
|Buffer 4
 +
|-
 +
|0,25 µl
 +
|Xba1
 +
|-
 +
|0,25 µl
 +
|Spe1-HF
 +
|-
 +
|0,2 µl
 +
|BSA(100x)
 +
|-
 +
|14,3 µl
 +
|ddH2O
 +
|}
 +
 +
Incubation: 37 °C; 1,5 h
 +
 +
A mastermix for 19 samples was made.
 +
 +
</div>
 +
 +
<div class="limonene">
 +
 +
=== Analytical gel electrophoresis of PCR1/pTUM104new, P144(PCR2)/pTUM104new, PCR1/pSB1C3 ===
 +
 +
'''Investigator:''' Lara
 +
 +
'''Aim of the experiment:''' Analytical gel electrophoresis of products from restriction digest.
 +
 +
[[File:TUM12_LS_analytgel2707_1.png]]
 +
 +
[[File:TUM12_LS_analytgel2707_2.png]]
 +
 +
</div>
 +
<div class="coumaryl">
 +
 +
===Small-Scale Yeast Transformation of 4CL, CHS, OMT and PAL- in pTUM104===
 +
'''Investigator:''' Daniela
 +
 +
'''Aim of the experiment:'''Transformation of P193 - P198 and P200 in Yeast
 +
 +
The protocol on page 13 of the pYES2 manual was used.
 +
 +
Only modifications are noted:
 +
 +
step 1: inoculate in 4 ml YPD medium
 +
 +
step 2: OD600 = 10 -> to determine a OD600 of 0.4 in 50 ml, 2.5 ml yeast suspension and 47.5 ml YPD medium were used (1:20 dilution)
 +
 +
steps 3 and 4: centrifugation for 5 min, 4 °C
 +
 +
step 5: room temperature was about 35 °C
 +
 +
step 6:
 +
 +
1 µg plasmid DNA
 +
 +
*P193: c = 110.7 ng/µl -> 9 µl
 +
*P194: c = 342.8 ng/µl -> 3 µl
 +
*P195: c = 423.6 ng/µl -> 2.4 µl 
 +
*P196: c = 92.1 ng/µl -> 11 µl
 +
*P197: c = 394.6 ng/µl -> 2.5 µl
 +
*P198: c = 183.4 ng/µl -> 5.5 µl 
 +
*P200: c = 464.5 ng/µl -> 2.2 µl
 +
 +
100 µg denatured sheared salmon sperm: the one from Simon was used -> 10 µl
 +
 +
step 8: incubation was carried out at 35 °C because the thermoblock did not achieve a lower temperature
 +
 +
The SCU- plates are incubated at 30°C over the weekend.
 +
 +
Colonies were grown on 2 plates (31.07.2012):
 +
CHS+ : 1 colony
 +
OMT+ : 3 colonies
 +
 +
-> repetition of transformation on plates with glucose!
 +
 +
</div>
 +
 +
== '''Saturday, July 28th''' ==
 +
 +
<div class="light_switchable_promoter">
 +
=== Miniprep of picked ''E.&nbsp;coli'' XL1-Blue transformated with ligation products of P126+PCR31, P151+PCR34, P123+PCR38 and P123+PCR34 ===
 +
</div>
 +
 +
== '''Sunday, July 29th''' ==
 +
<div class="coumaryl">
 +
===Picking of clones from PAL+ for a overnight culture===
 +
'''Investigator:''' Ingmar
 +
 +
'''Aim of the experiment:'''Inoculation of LB medium with clones picked from the transfomation of P183 (PAL+ (PCR32) in pYES (P175)) done on Monday, 23rd July in order to test on monday wheather the Ligation was successfull.
 +
'''Operationale sequence:'''
 +
*Two clones were picked an transferred to 6 ml LB medium containing 1:1000 Ampicillin.
 +
*Incubation overnight at 37°C, 180 rpm.
 +
</div>
 +
</div>
 +
 +
=Week 8=
 +
<!--
 +
<p class="limonene">'''Limonene (14 Experiments):'''</p>
 +
*
 +
 +
<p class="coumaryl">'''Xanthohumol (5 Experiments):'''</p>
 +
*
 +
 +
<p class="thaumatin">'''Thaumatin (2 Experiments):'''</p>
 +
* Media and plates for yeast transformation
 +
 +
<p class="constitutive_promoter">'''Constitutive Promoter (14 Experiments):'''</p>
 +
*
 +
 +
<p class="light_switchable_promoter">'''Light Switchable Promoter (16 Experiments):'''</p>
 +
*
 +
 +
<html><a class="WDetails" href="#Week_8" id="Week_8">Show Details ...</a></html>
 +
-->
 +
<div class="week" id="WWeek_8">
 +
== '''Monday, July 30th''' ==
 +
<div class="constitutive_promoter">
 +
===Extraction of Yeast genomic DNA for amplify TEF2-P, TEF1-T,ADH1-T===
 +
 +
'''Investigator''': Georg
 +
 +
'''Aim of Experiment:''' To get genomic DNA of yeast for amplify TEF2-P, TEF1-T,ADH1-T by PCR
 +
* 100 µl of overnight culture of S. cerevisiae is centrifuged 5 min (13400 rpm)
 +
* Remove supernatant and resuspend pellet in 100 µl 200 mM LiAC 1% SDS
 +
* Incubate for 5 min at 70 C
 +
* Precipitate DNA by adding 300µl 96% Ethanol and vortexing
 +
* Sedimentation for 3 min (13400 rpm)
 +
* Removal of supernatant and washing with 500 µl 70& EtOH + centrifuge
 +
* remove supernatant and solubilisate pellet in 100 ml 1x TE-buffer
 +
* Remove cell-parts by 1 min of centrifugation and move supernatant in new Eppi
 +
* Add 50 ml 7 M NH4AC-solution, let incubate at room-temperature for 5 min
 +
* Centrifuge for 10 min
 +
* Move supernatant to a new eppi
 +
* ADD 70 µl 7,5 M NH4OAc and 280 µl Isopropanol
 +
* Incubate 10 min at room-temperature and centrifuge 10 min
 +
* remove supernatant
 +
* wash pellet with 100µl 80% Ethanol, centrifuge for 10 min
 +
* remove supernatant
 +
* dry pelleted DNA, and resolubilsate in 1x TE-buffer
 +
( Extraction had to be done twice)
 +
</div>
 +
 +
<div class="constitutive_promoter">
 +
===Nanodrop of yeast genomic DNA to measuring the concentration===
 +
 +
'''Investigator:''' Georg
 +
 +
*S.C1: 51,2 ng/µl
 +
*S.C2: 24,6 ng/µl
 +
*S.C3: 41,3 ng/µl
 +
*S.C4: 45,9 ng/µl
 +
</div>
 +
<div class="light_switchable_promoter">
 +
=== Analytical digestion and gelelectrophoresis of the plasmids P221-P232 ===
 +
 +
'''Investigator: Saskia'''
 +
 +
'''Aim of the experiment:''' Analytical digest of P221-P223 with NdeI and BamHI and of P224-P232 with XbaI and AgeI-HF and analytical gelelectrophoresis .
 +
 +
'''Procedure:'''
 +
 +
* '''Analytical restriction digest and gelelectrophoresis of P221-P223 with NdeI and BamHI'''
 +
 +
* Reaction batch for each plasmid:
 +
{|cellspacing="0" border="1"
 +
|'''Reagent'''
 +
|'''Volume in µl'''
 +
|-
 +
|Tango Buffer 10x
 +
|4&nbsp;µl
 +
|-
 +
|BamHI (NEB)
 +
|0.5&nbsp;µl
 +
|-
 +
|NdeI (NEB)
 +
|0.5&nbsp;µl
 +
|-
 +
|Plasmid DNA
 +
|2.5&nbsp;µl
 +
|-
 +
|ddH2O
 +
|12.5&nbsp;µl
 +
|-
 +
|'''TOTAL'''
 +
|'''20&nbsp;µl'''
 +
|}
 +
 +
* Incubation at 37&nbsp;°C for 1&nbsp;h 20&nbsp;min.
 +
 +
* Analytical gelelectrophoresis  (1%) at 90&nbsp;V for 40-45 min.
 +
 +
* '''Analytical restriction digest and gelelectrophoresis of P224-P232 with XbaI and AgeI-HF'''
 +
* Reaction batch for each plasmid:
 +
{|cellspacing="0" border="1"
 +
|'''Reagent'''
 +
|'''Volume in µl'''
 +
|-
 +
|NEBuffer4 10x
 +
|2&nbsp;µl
 +
|-
 +
|BSA 100x
 +
|0.2&nbsp;µl
 +
|-
 +
|XbaI (Fermentas)
 +
|0.25&nbsp;µl
 +
|-
 +
|AgeI-HF (Fermentas)
 +
|0.25&nbsp;µl
 +
|-
 +
|Plasmid DNA
 +
|2.5&nbsp;µl
 +
|-
 +
|ddH2O
 +
|14.8&nbsp;µl
 +
|-
 +
|'''TOTAL'''
 +
|'''20&nbsp;µl'''
 +
|}
 +
 +
* Incubation at 37&nbsp;°C for 1&nbsp;h 20&nbsp;min.
 +
 +
* Analytical gelelectrophoresis  (1%) at 90&nbsp;V for 40-45 min.
 +
 +
* Order of gel-pockets:
 +
{|cellspacing="0" border="1"
 +
|1&nbsp;kbp ladder
 +
|P221