Team:Marburg SYNMIKRO/Notebook
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- | < | + | {{:Team:Marburg_SYNMIKRO:Template:Header}} |
+ | <html> | ||
+ | <style> | ||
+ | |||
+ | #toc ul { | ||
+ | display:none; | ||
+ | } | ||
+ | |||
+ | .editsection{ | ||
+ | display:none; | ||
+ | } | ||
+ | |||
+ | .fixcon{ | ||
+ | position:relative; | ||
+ | } | ||
+ | |||
+ | .toggleall{ | ||
+ | position:absolute; | ||
+ | top:-60px; | ||
+ | right:5px; | ||
+ | font-size: 10pt; | ||
+ | color: green; | ||
+ | cursor:pointer; | ||
+ | } | ||
+ | |||
+ | .fixtoggle{ | ||
+ | position:absolute; | ||
+ | top:-38px; | ||
+ | right:5px; | ||
+ | background-color:rgba(255,255,255,0.01); | ||
+ | font-size: 10pt; | ||
+ | color: green; | ||
+ | cursor:pointer; | ||
+ | background-image:url('http://web383.mis60.de/inprogress/down-arrow.png'); | ||
+ | background-repeat:no-repeat; | ||
+ | background-position:right; | ||
+ | width:960px; | ||
+ | height:18px; | ||
+ | } | ||
+ | |||
+ | .smalldate{ | ||
+ | position:absolute; | ||
+ | top:-38px; | ||
+ | left:115px; | ||
+ | background-color:transparent; | ||
+ | font-size: 14pt; | ||
+ | color: grey; | ||
+ | } | ||
+ | |||
+ | </style> | ||
+ | <script type="text/javascript"> | ||
+ | function toggledisplay (id){ | ||
+ | if (document.getElementById) { | ||
+ | var mydiv = document.getElementById(id); | ||
+ | mydiv.style.display = (mydiv.style.display=='block'?'none':'block'); | ||
+ | } | ||
+ | } | ||
+ | function togglearrow (id){ | ||
+ | if (document.defaultView.getComputedStyle(id,null).getPropertyValue('background-color')!='transparent') { | ||
+ | id.style.backgroundColor='transparent'; | ||
+ | id.style.backgroundImage='url(http://web383.mis60.de/inprogress/up-arrow.png)' | ||
+ | } | ||
+ | else{ | ||
+ | id.style.backgroundColor='rgba(255,255,255,0.01)'; | ||
+ | id.style.backgroundImage='url(http://web383.mis60.de/inprogress/down-arrow.png)' | ||
+ | } | ||
+ | } | ||
+ | </script> | ||
+ | </html> | ||
<html> | <html> | ||
- | <div | + | <div class="conboxb"> |
- | <div | + | </html> |
- | + | = Week 1 = | |
+ | <html> | ||
+ | <div class="fixcon"> | ||
+ | <div class="toggleall" onClick="javascript:toggledisplay('klapp1');javascript:toggledisplay('klapp2');javascript:toggledisplay('klapp3');javascript:toggledisplay('klapp4');javascript:toggledisplay('klapp5');javascript:toggledisplay('klapp6');javascript:toggledisplay('klapp7');javascript:toggledisplay('klapp8');javascript:toggledisplay('klapp9');javascript:toggledisplay('klapp10');javascript:toggledisplay('klapp11');" return false">[all]</div> | ||
+ | <div class="fixtoggle" onClick="javascript:toggledisplay('klapp1'); togglearrow(this);" return false"></div> | ||
+ | <div class="smalldate">09.07. - 15.07.2012</div> | ||
</div> | </div> | ||
- | <div | + | <div class="hiddenifjs" id="klapp1"> |
- | + | </html> | |
+ | |||
+ | |||
+ | ==09.07.2012== | ||
+ | ; Inoculation | ||
+ | : ''E. coli'' strain C600 Mucts62 in dYT-medium | ||
+ | |||
+ | ==10.07.2012== | ||
+ | ; Isolation of chromosomal DNA | ||
+ | : ''E. coli'' strain C600 Mucts62 (lysogenic for temperature sensitive phage Mu ''c''ts62) | ||
+ | ; Inoculation | ||
+ | : ''E. coli'' strain DH5α and TOP10 in dYT-medium | ||
+ | |||
+ | ==11.07.2012== | ||
+ | ; Preparation of chemically competent ''E. coli'' cells | ||
+ | : TOP10 | ||
+ | |||
+ | ==12.07.2012== | ||
+ | ; Transformation | ||
+ | : GFP, GFP_fusion, CFP, mRFP, RBS, P108, P100, Plac with ''E.coli'' TOP10 | ||
+ | |||
+ | {|class="wikitable" | ||
+ | !Name!!Biobrick!!Plate!!Well!!Backbone!!Resistance | ||
+ | |- | ||
+ | |GFP||[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]||1||14K||pSB1A2||Ampicillin | ||
+ | |- | ||
+ | |GFP_fusion||[http://partsregistry.org/Part:BBa_K125500 BBa_K125500]||3||2P||pSB1A2||Ampicillin | ||
+ | |- | ||
+ | |CFP||[http://partsregistry.org/Part:BBa_E0020 BBa_E0020]||1||6A||pSB1A2||Ampicillin | ||
+ | |- | ||
+ | |mRFP||[http://partsregistry.org/Part:BBa_E1010 BBa_E1010]||1||18F||pSB2K3||Kanamycin | ||
+ | |- | ||
+ | |RBS||[http://partsregistry.org/Part:BBa_J61100 BBa_J61100]||1||5J||pSB1A2||Ampicillin | ||
+ | |- | ||
+ | |P108||[http://partsregistry.org/Part:BBa_J23108 BBa_J23108]||2||2E||BBa_J61002||Ampicillin | ||
+ | |- | ||
+ | |P100||[http://partsregistry.org/Part:BBa_J23100 BBa_J23100]||1||18C||BBa_J61002||Ampicillin | ||
+ | |- | ||
+ | |PLac||[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]||2||11P||pSB2K3||Kanamycin | ||
+ | |} | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <html> | ||
</div> | </div> | ||
- | < | + | </html> |
- | + | <html> | |
</div> | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | |||
+ | = Week 2 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp2'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate">16.07. - 22.07.2012 </div> | ||
</div> | </div> | ||
+ | <div class="hiddenifjs" id="klapp2"> | ||
</html> | </html> | ||
- | + | ==16.07.2012== | |
+ | ; PCR | ||
+ | : AmiC (primer: [[Team:Marburg_SYNMIKRO/Primer#MS1|MS1]] + [[Team:Marburg_SYNMIKRO/Primer#MS2|MS2]]) | ||
+ | : HU (primer: [[Team:Marburg_SYNMIKRO/Primer#MS3|MS3]] + [[Team:Marburg_SYNMIKRO/Primer#MS4|MS4]]) | ||
+ | : GroES (primer: [[Team:Marburg_SYNMIKRO/Primer#MS5|MS5]] + [[Team:Marburg_SYNMIKRO/Primer#MS6|MS6]]) | ||
+ | : GixR (primer: [[Team:Marburg_SYNMIKRO/Primer#MS7|MS7]] + [[Team:Marburg_SYNMIKRO/Primer#MS8|MS8]]) | ||
+ | : Enhancer (primer: [[Team:Marburg_SYNMIKRO/Primer#MS9|MS9]] + [[Team:Marburg_SYNMIKRO/Primer#MS10|MS10]]) | ||
+ | : ⇒ template-DNA: chromosomal DNA, denaturation: 30 sec at 95°C; annealing: 30 sec at 60°C; elongation: 30 sec at 72°C; 35 cycles | ||
- | + | ; Inoculation | |
- | + | : GFP | |
- | + | : GFP_fusion | |
- | + | : CFP | |
- | + | : mRFP | |
- | + | : RBS | |
- | + | : P108 | |
- | + | : LacIQ | |
- | + | : P100 | |
- | + | ||
- | + | ||
+ | ==17.07.2012== | ||
+ | ; Plasmid preparation | ||
+ | :GFP | ||
+ | :GFP_fusion | ||
+ | :CFP | ||
+ | :mRFP | ||
+ | :RBS | ||
+ | :P108 | ||
+ | :LacIq | ||
+ | :P100 | ||
+ | ==18.07.2012== | ||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel1.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:enhancer, 2:GroES, 3:AmiC, 4:HU, 5:GixR, 6:GFP, 7:GFP-Fusion, 8:CFP, 9:mRFP, 10:RBS, 11:LacIQ, 12:P108, 13:P100, 14:λ-DNA (PCR 18.07.2012)]] | ||
+ | |||
+ | ; DNA Gel extraction | ||
+ | : Enhancer | ||
+ | : GroES | ||
+ | : HU | ||
+ | |||
+ | ; GixR Hybridization | ||
+ | * 5 µl primer [[Team:Marburg_SYNMIKRO/Primer#MS7|MS7]] (100 µM) + 5 µl primer [[Team:Marburg_SYNMIKRO/Primer#MS8|MS8]] (100 µM) | ||
+ | ** 5 min 99°C | ||
+ | ** over night, room temperature | ||
+ | ** -80°C | ||
+ | |||
+ | ==19.07.2012== | ||
+ | ;Fill-in reaction (primer extension) | ||
+ | : Enhancer | ||
+ | |||
+ | ; Restriction | ||
+ | : Enhancer (EcoRI/PstI) | ||
+ | |||
+ | ; PCR | ||
+ | : AmiC (primer: [[Team:Marburg_SYNMIKRO/Primer#MS1|MS1]] + [[Team:Marburg_SYNMIKRO/Primer#MS2|MS2]]) | ||
+ | |||
+ | |||
+ | : ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 85 °C, elongation: 36 sec; 72 °C, 35 cycles, 1) 2 µl DMSO; 1 µl chrom. DNA | 2) 2 µl DMSO; 5 µl chrom. DNA | 3) without DMSO; 1 µl chrom. DNA | 4) without DMSO; 5 µl chrom. DNA | 5) 2 µl DMSO; without chrom. DNA | ||
+ | |||
+ | ; Test restriction digest | ||
+ | : 7) CFP (EcoRI/PstI) | ||
+ | : 8) GFP (EcoRI/PstI) | ||
+ | : 9) GFP-fusion (EcoRI/PstI) | ||
+ | : 10) mRFP (EcoRI/PstI) | ||
+ | : ⇒ all negative | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel2.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-5:AmiC PCR conditions (see above), 6-9: control digest (E/P), 6:GFP, 7:GFP-Fusion, 8:CFP, 9:mRFP (PCR 19.07.2012)]] | ||
+ | |||
+ | ; Control digest | ||
+ | : CFP (EcoRI/PstI) | ||
+ | : GFP (EcoRI/PstI) | ||
+ | : GFP-fusion (EcoRI/PstI) | ||
+ | : mRFP (EcoRI/PstI) | ||
+ | : ⇒ restriction over night | ||
+ | |||
+ | ==20.07.2012== | ||
+ | ; 1% Agarose gel from the control digest | ||
+ | [[File:Syn_MR_Gel3.jpg|thumb|400px|center|M:1kb gene ruler plus, 1:GFP, 2: GFP-Fusion, 3: CFP, 4: mRFP (control digest 18.07.2012)]] | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | = Week 3 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp3'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate">23.07. - 29.07.2012</div> | ||
+ | </div> | ||
+ | <div class="hiddenifjs" id="klapp3"> | ||
+ | </html> | ||
+ | |||
+ | ==26.07.2012== | ||
+ | ; Restriction | ||
+ | : pSB1T3 (XbaI/SpeI and DpnI) | ||
+ | : pSB1A3 (XbaI/SpeI and DpnI) | ||
+ | : pSB1K3 (XbaI/SpeI and DpnI) | ||
+ | : pSB1C3 (XbaI/SpeI and DpnI) | ||
+ | |||
+ | ; Ligation | ||
+ | : GixR + pSB1A3 | ||
+ | : Enhancer + pSB1K3 | ||
+ | : cyclisation of pSB1C3, pSB1K3, pSB1A3, pSB1T3 (couldn’t work) | ||
+ | |||
+ | ==27.07.2012== | ||
+ | Our lab-dragon "Hugo" is born | ||
+ | [[File:Syn_MR_Hugo.jpg|frameless|border|650px|center]] | ||
+ | |||
+ | |||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | = Week 4 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp4'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate">30.07. - 05.08.2012</div> | ||
+ | </div> | ||
+ | <div class="hiddenifjs" id="klapp4"> | ||
+ | </html> | ||
+ | |||
+ | |||
+ | ==30.07.2012== | ||
+ | |||
+ | ; PCR | ||
+ | : CFP (primer: [[Team:Marburg_SYNMIKRO/Primer#MS16|MS16]] + [[Team:Marburg_SYNMIKRO/Primer#MS17|MS17]]) ⇒ template-DNA: [http://partsregistry.org/Part:BBa_E0020 BBa_E0020] | ||
+ | : ⇒ positive | ||
+ | : mRFP (primer: [[Team:Marburg_SYNMIKRO/Primer#MS14|MS14]] + [[Team:Marburg_SYNMIKRO/Primer#MS15|MS15]]) ⇒ template-DNA: [http://partsregistry.org/Part:BBa_E1010 BBa_E1010] | ||
+ | : ⇒ positive | ||
+ | : Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS11|MS11]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]]) ⇒ template-DNA: chromosomal DNA (that was wrong) | ||
+ | : RBS-Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]] + [[Team:Marburg_SYNMIKRO/Primer#MS12|MS12]]) ⇒ template-DNA: chromosomal DNA (that was wrong) | ||
+ | : ⇒ annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles | ||
+ | |||
+ | ; Transformation | ||
+ | : all vector-backbone-ligations | ||
+ | : Enhancer (K3) | ||
+ | : GixR (A3) | ||
+ | |||
+ | ==31.07.2012== | ||
+ | ; Result: transformation | ||
+ | : pSB1A3: 3 colonies | ||
+ | : pSB1K3: 60 colonies | ||
+ | : pSB1C3: 9 colonies | ||
+ | : pSB1T3: 0 colonies | ||
+ | : Enhancer (K3): 0 colonies | ||
+ | : GixR (A3): 0 colonies | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel4.jpg|thumb|400px|center|M:Marker, 1: Gix, 2-6: AmiC (PCR 16.07.2012)]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel5.jpg|thumb|300px|center|M:1kb gene ruler plus, 1: AmiC(PCR 16.07.2012)]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel6.jpg|thumb|300px|center|M:1kb gene ruler plus, 1: RBS-Gin from 30.07, 2:AmiC from 16.07., 3:Gin from 30.07., 4:CFP from 30.07., 5:mRFP from 30.07., 6: control (PCR 30.07.2012)]] | ||
+ | |||
+ | |||
+ | ; Resuspension of a biobrick | ||
+ | : [http://partsregistry.org/Part:BBa_B0010 BBa_B0010](Terminator) | ||
+ | |||
+ | ; DNA Gel extraction | ||
+ | : CFP | ||
+ | : mRFP | ||
+ | |||
+ | ; PCR | ||
+ | : AmiC (primer: [[Team:Marburg_SYNMIKRO/Primer#MS1|MS1]] + [[Team:Marburg_SYNMIKRO/Primer#MS2|MS2]]) | ||
+ | : RBS-Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS12|MS12]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]]) | ||
+ | : Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS11|MS11]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]]) | ||
+ | : ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 40 sec; 72 °C, 35 cycles | ||
+ | |||
+ | ; Restriction | ||
+ | : pSB1A3 (EcoRI/PstI) | ||
+ | : CFP (EcoRI/PstI) | ||
+ | : mRFP (EcoRI/PstI) | ||
+ | |||
+ | ; Transformation | ||
+ | : pSB1A3 | ||
+ | : pSB1C3 | ||
+ | : pSB1T3 | ||
+ | : Terminator | ||
+ | : GixR | ||
+ | : Enhancer | ||
+ | |||
+ | ; Inoculation | ||
+ | : pSB1A3 | ||
+ | : pSB1C3 | ||
+ | : pSB1K3 | ||
+ | |||
+ | ; Ligation | ||
+ | : pSB1A3 + CFP | ||
+ | : pSB1A3 + mRFP | ||
+ | |||
+ | ; GixR Hybridization | ||
+ | * 5 µl primer [[Team:Marburg_SYNMIKRO/Primer#MS7|MS7]] (100 µM) + 5 µl primer [[Team:Marburg_SYNMIKRO/Primer#MS8|MS8]] (100 µM) | ||
+ | ** 5 min 99°C | ||
+ | ** over night, room temperature | ||
+ | ** -80°C | ||
+ | |||
+ | ; DNA Gel extraction | ||
+ | : AmiC | ||
+ | |||
+ | ; Ligation | ||
+ | : AmiC + pSV2 (suicide vector) | ||
+ | |||
+ | |||
+ | ==01.08.2012== | ||
+ | |||
+ | ; Result: transformation | ||
+ | : Only BBa_B0010 colonies present | ||
+ | |||
+ | ; Plasmid preparation | ||
+ | : pSB1A3 ⇒ only clone 1, 3 Positive | ||
+ | : pSB1C3 ⇒ all negative | ||
+ | : pSB1K3 ⇒ all negative | ||
+ | |||
+ | ; Ligation | ||
+ | : GixR hyb. + pJET2_1 | ||
+ | : ⇒ over blunt ends | ||
+ | |||
+ | ; Restriction | ||
+ | : GixR hyb. (EcoRI/PstI) | ||
+ | : pSB1C3 (EcoRI/PstI) | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel7.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: RBS-Gin (+DMSO), 2:RBS-Gin (-DMSO), 3:Gin (+DMSO), 4:Gin (-DMSO), 5: control (PCR 31.07.2012)]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel8.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: AmiC (+DMSO), 2:AmiC (-DMSO), (PCR 31.07.2012)]] | ||
+ | |||
+ | ; DNA Gel extraction | ||
+ | : RBS-Gin | ||
+ | : Gin | ||
+ | : AmiC | ||
+ | |||
+ | ; Test restriction | ||
+ | : pSB1A3 (EcoRI) | ||
+ | |||
+ | ; Restriction | ||
+ | : RBS-Gin (EcoRI/PstI) | ||
+ | : Gin (EcoRI/PstI) | ||
+ | : AmiC (EcoRI/PstI) | ||
+ | : pSB1C3 (EcoRI/PstI) | ||
+ | : pSB1K3 (EcoRI/PstI) | ||
+ | : pSB1T3 (EcoRI/PstI) | ||
+ | |||
+ | ; Ligation | ||
+ | : cyclisation of pSB1T3 | ||
+ | |||
+ | ;Fill-in reaction (primer extension) | ||
+ | : Enhancer | ||
+ | |||
+ | ; Ligation | ||
+ | : Enhancer and GixR in pSV2 | ||
+ | : Enhancer in pSB1K3 | ||
+ | : GixR in pSB1C3 | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel9.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-3: pSB1A3, 4-7:pSB1C3, 8-11: pSB1K3 (Control digest 01.08.2012)]] | ||
+ | |||
+ | |||
+ | : pSB1A3 c2 | ||
+ | : pSB1C3 c4 | ||
+ | : pSB1K3 c3 | ||
+ | |||
+ | ; Transformation | ||
+ | : pSB1A3 religation | ||
+ | : CFP in A3 | ||
+ | : mRFP in A3 | ||
+ | : AmiC in pJET | ||
+ | : GixR in pJET | ||
+ | : Enhancer in pJET | ||
+ | : GixR in C3 | ||
+ | : Enhancer in K3 | ||
+ | |||
+ | ; Ligation | ||
+ | : AmiC + pSB1T3 | ||
+ | : RBS-Gin + pSB1K3 | ||
+ | : Gin + pSB1K3 | ||
+ | |||
+ | |||
+ | ==02.08.2012== | ||
+ | |||
+ | ; Resuspension of a biobrick | ||
+ | : pSB3C5 | ||
+ | : ⇒ for ccdB | ||
+ | |||
+ | ; Plasmid preparation | ||
+ | : BBa_B0010 (terminator) | ||
+ | |||
+ | ; Test restriction | ||
+ | : Terminator c1,2,3,4 (EcoRI/PstI) | ||
+ | |||
+ | |||
+ | ; 2% Agarose gel | ||
+ | [[File:Syn_MR_Gel10.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-4: Terminator, M2:Middle Range gene ruler (Control digest 01.08.2012)]] | ||
+ | |||
+ | ; Transformation | ||
+ | : pSB1T3 recycled | ||
+ | : AmiC in T3 | ||
+ | : Gin-RBS in K3 | ||
+ | : Gin in K3 | ||
+ | : pSB3C5 linear (false) | ||
+ | |||
+ | |||
+ | ==03.08.2012== | ||
+ | |||
+ | ; Plasmid preparation | ||
+ | : Enhancer in pJET | ||
+ | |||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | = Week 5 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp5'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate">06.08. - 12.08.2012</div> | ||
+ | </div> | ||
+ | <div class="hiddenifjs" id="klapp5"> | ||
+ | </html> | ||
+ | |||
+ | |||
+ | ==06.08.2012== | ||
+ | |||
+ | ; Resuspension and transformation of a biobrick | ||
+ | : BBa_J04450 in pSB1A3 | ||
+ | : BBa_J04450 in pSB1C3 | ||
+ | : BBa_J04450 in pSB1K3 | ||
+ | : BBa_J04450 in pSB1T3 | ||
+ | |||
+ | ; Restriction | ||
+ | : GFP-fusion (EcoRI/SpeI) | ||
+ | : Terminator (XbaI/PstI) | ||
+ | : Promoter BBa_J23100 (EcoRI/SpeI) | ||
+ | : Promoter BBa_J23108 (EcoRI/SpeI) | ||
+ | : RBS (XbaI/PstI) | ||
+ | |||
+ | ; Ligation | ||
+ | : RBS-Gin in T3 | ||
+ | : Gin in T3 | ||
+ | : GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3 | ||
+ | : Promoter BBa_J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3 | ||
+ | : Promoter BBa_J23100 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3 | ||
+ | |||
+ | ; GixR Hybridization | ||
+ | * 1(3) µl primer [[Team:Marburg_SYNMIKRO/Primer#MS7|MS7]]7 (100 µM) + 1(3) µl primer [[Team:Marburg_SYNMIKRO/Primer#MS8|MS8]] (100 µM) + T4-Ligasebuffer | ||
+ | ** 5 min 95°C | ||
+ | ** over night, room temperature | ||
+ | ** -80°C | ||
+ | |||
+ | ; Transformation | ||
+ | : GixR in C3 | ||
+ | : Gin in T3 | ||
+ | : RBS-Gin in T3 | ||
+ | : mRFP in A3 | ||
+ | : CFP in A3 | ||
+ | |||
+ | ==07.08.2012== | ||
+ | |||
+ | ; Transformation | ||
+ | : GFP-fusion | ||
+ | : CFP | ||
+ | : J23108-RBS | ||
+ | : J23100-RBS | ||
+ | |||
+ | ; Plasmid preparation | ||
+ | : GixR 1,2,3 | ||
+ | |||
+ | ; Restriction | ||
+ | : GixR hyb. 1,2,3 (EcoRI/PstI) | ||
+ | : pSB1C3 (EcoRI/PstI) | ||
+ | |||
+ | ; Ligation | ||
+ | : GixR hyb. 1,2,3 in pJET | ||
+ | : GixR hyb. 1,2,3 in C3 | ||
+ | |||
+ | |||
+ | ; Transformation | ||
+ | : GixR hyb. 1,2,3 in pJET | ||
+ | : GixR hyb. 1,2,3 in C3 | ||
+ | |||
+ | ; Test restriction | ||
+ | : pSB3C5 (XbaI) | ||
+ | : pJET (XbaI) | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel11.jpg|thumb|700px|center|M:1kb gene ruler plus, 1-4: pSB3C5, 5-8:pSB3C5, 9-12: Enhancer-pJET (Control digest 07.08.2012)]] | ||
+ | |||
+ | : ⇒ positive: pSB3C5 3,4,7,8,; Enhancer-pJET 9-12 | ||
+ | ==08.08.2012== | ||
+ | |||
+ | ; Result transformation | ||
+ | : GFP-fusion (20 clones) | ||
+ | : J23108-RBS (20 clones) | ||
+ | : J23100-RBS (20 clones) | ||
+ | : GixR hyb. 1,2,3 in pJET (10 clones) | ||
+ | : GixR hyb. 1,2,3 in C3 (10 clones) | ||
+ | : CFP (5 clones) | ||
+ | |||
+ | ; Restriktion | ||
+ | : Enhancer in pJET (EcoRI/PstI) | ||
+ | : pSB1C3 (EcoRI/PstI) | ||
+ | |||
+ | ; 2% Agarose gel | ||
+ | [[File:Syn_MR_Gel12.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: Enhancer, 2:pSB1C3, (Control digest 08.08.2012)]] | ||
+ | |||
+ | ; DNA Gel extraction | ||
+ | : Enhancer | ||
+ | : pSB1C3 | ||
+ | |||
+ | ; Ligation | ||
+ | : Enhancer 1,2 in C3 | ||
+ | |||
+ | ; Transformation | ||
+ | : Enhancer 1,2 in C3 | ||
+ | : C3 | ||
+ | : mRFP | ||
+ | |||
+ | ==09.08.2012== | ||
+ | |||
+ | ; Result transformation | ||
+ | : GFP-fusion ⇒ positive | ||
+ | : J23108-RBS ⇒ positive | ||
+ | : J23100-RBS ⇒ positive | ||
+ | : CFP ⇒ positive | ||
+ | : GixR in C3 c1 ⇒ negative | ||
+ | : GixR in C3 c2 ⇒ negative | ||
+ | : GixR in C3 c3 ⇒ negative | ||
+ | |||
+ | ; Plasmid preparation | ||
+ | : GFP-fusion | ||
+ | : J23108-RBS | ||
+ | : J23100-RBS | ||
+ | : CFP | ||
+ | |||
+ | ; Test restriction | ||
+ | : GFP-fusion (EcoRI/PstI) | ||
+ | : J23108-RBS (EcoRI) | ||
+ | : J23100-RBS (EcoRI) | ||
+ | : CFP (EcoRI/PstI) | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel13.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-18: GFP-Fusion - Terminator, (Control digest 09.08.2012)]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel14.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-18: P108-RBS, (Control digest 09.08.2012)]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel15.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-18: P100-RBS, (Control digest 09.08.2012)]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel16.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-2: GFP-Fusion - Terminator, 3-4: P108-RBS, 5-6: P100-RBS, 7-11: CFP (Control digest 09.08.2012)]] | ||
+ | |||
+ | |||
+ | ; Inoculation | ||
+ | : GixR in C3 | ||
+ | : Gin in T3 | ||
+ | : Gin-RBS in T3 | ||
+ | : pSB1C3 | ||
+ | : Enhancer in C3 | ||
+ | : mRFP in A3 | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | = Week 6 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp6'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate">13.08. - 19.08.2012</div> | ||
+ | </div> | ||
+ | <div class="hiddenifjs" id="klapp6"> | ||
+ | </html> | ||
+ | |||
+ | |||
+ | == 13.08.12 == | ||
+ | |||
+ | ; PCR | ||
+ | : GroES (primer: [[Team:Marburg_SYNMIKRO/Primer#MS5|MS5]] + [[Team:Marburg_SYNMIKRO/Primer#MS6b|MS6b]]) | ||
+ | : HU (primer: [[Team:Marburg_SYNMIKRO/Primer#MS3|MS3]] + [[Team:Marburg_SYNMIKRO/Primer#MS4b|MS4b]]) | ||
+ | : ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel17.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: HU (+DMSO), 2: HU (-DMSO), 3: GroES (+DMSO), 4: GroES (-DMSO), (PCR 13.08.2012)]] | ||
+ | |||
+ | ; Transformation | ||
+ | : LacIQ (BBa_I14032) | ||
+ | |||
+ | |||
+ | == 14.08.12 == | ||
+ | |||
+ | ; Restriction | ||
+ | : pSB1K3 (EcoRI/PstI) | ||
+ | : Enhancer 3,4 (EcoRI/ PstI) | ||
+ | : GFP-fusion (EcoRI/SpeI) | ||
+ | : Terminator (XbaI/PstI) | ||
+ | |||
+ | ; Test restriction | ||
+ | : RBS-Gin in T3 (EcoRI/ PstI) | ||
+ | |||
+ | ; Ligation | ||
+ | : GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3 (EcoRI/PstI) | ||
+ | : Promotor J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3 (EcoRI/PstI) | ||
+ | : Enhancer 3,4 (EcoRI/ PstI) + pSB1K3 (EcoRI/PstI) | ||
+ | |||
+ | ; Transformation | ||
+ | : GFP-fusion-Terminator in pSB1K3 | ||
+ | : Promotor-RBS in pSB1K3 | ||
+ | : Enhancer 3 in pSB1K3 | ||
+ | : Enhancer 4 in pSB1K3 | ||
+ | : GixR in pJET | ||
+ | |||
+ | |||
+ | == 15.08.2012 == | ||
+ | |||
+ | ; PCR | ||
+ | : GroES (primer: [[Team:Marburg_SYNMIKRO/Primer#MS5|MS5]] + [[Team:Marburg_SYNMIKRO/Primer#MS6b|MS6b]]) | ||
+ | : HU (primer: [[Team:Marburg_SYNMIKRO/Primer#MS3|MS3]] + [[Team:Marburg_SYNMIKRO/Primer#MS4b|MS4b]]) | ||
+ | : ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C, 35 cycles | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel18.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: HU (-DMSO), 2: HU (+DMSO), 3: GroES (-DMSO), 4: GroES (+DMSO), (PCR 15.08.2012)]] | ||
+ | |||
+ | ; DNA Gel extraction | ||
+ | : HU 1,2 | ||
+ | |||
+ | ; Resuspension of a biobrick | ||
+ | : BBa_J04450 in pSB1A3 | ||
+ | : BBa_J04450 in pSB1T3 | ||
+ | : BBa_J04450 in pSB1C3 | ||
+ | : BBa_J04450 in pSB1K3 | ||
+ | |||
+ | |||
+ | ; Transformation | ||
+ | : BBa_J04450 in pSB1A3 | ||
+ | : BBa_J04450 in pSB1T3 | ||
+ | : BBa_J04450 in pSB1C3 | ||
+ | : BBa_J04450 in pSB1K3 | ||
+ | |||
+ | ; Test restriction | ||
+ | : pSB1T3 (EcoRI/PstI and DpnI) | ||
+ | : pSB1K3 (EcoRI/PstI and DpnI) | ||
+ | : pSB1C3 (EcoRI/PstI and DpnI) | ||
+ | : pSB1A3 (EcoRI/PstI and DpnI) | ||
+ | |||
+ | |||
+ | == 16.08.2012 == | ||
+ | |||
+ | ; PCR | ||
+ | : GroES (primer: [[Team:Marburg_SYNMIKRO/Primer#MS5|MS5]] + [[Team:Marburg_SYNMIKRO/Primer#MS6b|MS6b]]) | ||
+ | : ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel19.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: GroES (-DMSO), 2: GroES (+DMSO), (PCR 16.08.2012)]] | ||
+ | |||
+ | ; Plasmid preparation | ||
+ | : P-RBS | ||
+ | |||
+ | ; Test restriction | ||
+ | : P-RBS (EcoRI/PstI) | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel20.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: control, 2: P108-RBS, (control digest 16.08.2012)]] | ||
+ | |||
+ | ; Restriction | ||
+ | : Enhancer 3,4 (EcoRI/ PstI) | ||
+ | : HU (EcoRI/ PstI) | ||
+ | : AmiC (EcoRI/ PstI) | ||
+ | : Promoter, GFP (EcoRI/SpeI) | ||
+ | : RBS; Terminator (XbaI/PstI) | ||
+ | |||
+ | ; Ligation | ||
+ | : HU, AmiC in pSB1A3 | ||
+ | : Enhancer 3,4 in pSB1K3 | ||
+ | : P-RBS in pSB1K3 | ||
+ | : GFP fusion-Terminator in pSB1K3 | ||
+ | |||
+ | ; DNA Gel extraction | ||
+ | : GroES 1, P-RBS | ||
+ | |||
+ | ; Transformation | ||
+ | : AmiC | ||
+ | : HU | ||
+ | : Enhancer 3,4, 3 alt,4 alt | ||
+ | |||
+ | ; Inoculation | ||
+ | : BBa_J04450 in pSB1A3 | ||
+ | : BBa_J04450 in pSB1T3 | ||
+ | : BBa_J04450 in pSB1C3 | ||
+ | : BBa_J04450 in pSB1K3 | ||
+ | : AmiC | ||
+ | : P-RBS | ||
+ | |||
+ | == 17.08.2012 == | ||
+ | |||
+ | ; Restriction | ||
+ | : GroES (EcoRI/SpeI) | ||
+ | |||
+ | ; Ligation | ||
+ | : GroES in pSB1C3 | ||
+ | |||
+ | ; Transformation | ||
+ | : GroES in pSB1C3 | ||
+ | |||
+ | ; Test restriction | ||
+ | : P-RBS (EcoRI/PstI) | ||
+ | : BBa_J04450 in pSB1A3 (EcoRI/PstI) | ||
+ | : BBa_J04450 in pSB1T3 (EcoRI/PstI) | ||
+ | : BBa_J04450 in pSB1C3 (EcoRI/PstI) | ||
+ | : BBa_J04450 in pSB1K3 (EcoRI/PstI) | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel21.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-4: BBa_J04450 in pSB1A3, 5-8: BBa_J04450 in pSB1K3 P108-RBS, 9-12: BBa_J04450 in pSB1C3, (control digest 17.08.2012)]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel22.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-4: P-RBS (E/P), 5-8: P-RBS (RsaI), 9-12: BBa_J04450 in pSB1T3, (control digest 17.08.2012)]] | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | = Week 7 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp7'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate">20.08. - 26.08.2012</div> | ||
+ | </div> | ||
+ | <div class="hiddenifjs" id="klapp7"> | ||
+ | </html> | ||
+ | |||
+ | |||
+ | == 20.08.2012 == | ||
+ | |||
+ | ; PCR | ||
+ | : Backbone (primer [[Team:Marburg_SYNMIKRO/Primer#MS24|MS24]] + [[Team:Marburg_SYNMIKRO/Primer#MS25|MS25]]) | ||
+ | : ⇒ template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | [[File:Syn_MR_Gel23.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: pSB1A3, 2: pSB1T3, 3: pSB1C3, 4: pSB1K3 (PCR 20.08.2012)]] | ||
+ | |||
+ | ; Transformation | ||
+ | : BBa_JH023 | ||
+ | |||
+ | ; Inoculation | ||
+ | : GroES | ||
+ | : P-RBS | ||
+ | : GFP-T | ||
+ | : Enhancer 3,4 | ||
+ | : AmiC | ||
+ | : P-RBS alt | ||
+ | : GFP-T alt | ||
+ | |||
+ | == 21.08.2012 == | ||
+ | |||
+ | ; Results | ||
+ | : colonies of BBa_JH023 | ||
+ | : inoculations except AmiC and P-RBS alt successful | ||
+ | |||
+ | ; PCR | ||
+ | : pSB1A3-BBa_J04450 (template DNA) | ||
+ | : pSB1T3-BBa_J04450 (template DNA) | ||
+ | : pSB1C3-BBa_J04450 (template DNA) | ||
+ | : pSB1K3-BBa_J04450 (template DNA) | ||
+ | : used primer --> [[Team:Marburg_SYNMIKRO/Primer#MS24|MS24]] + [[Team:Marburg_SYNMIKRO/Primer#MS25|MS25]] | ||
+ | : initial denaturation 98°C - 5min | ||
+ | : denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times | ||
+ | : 72°C - 5min --> 4°C - 8 | ||
+ | |||
+ | ; miniprep | ||
+ | : GroES, Enh 3/4 (+old), P-RBS (+old), GFP-T (+old) | ||
+ | |||
+ | ; Test restriction | ||
+ | : of miniprep | ||
+ | : over night, 37°C | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel24.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:pSB1A3, 2:pSB1K3, 3:pSB1C3, 4:pSB1T3, 5: negative control, (PCR 21.08.2012)]] | ||
+ | |||
+ | ; Inoculation | ||
+ | : E.coli BBa_I14032 | ||
+ | : over night, 37°C | ||
+ | |||
+ | |||
+ | == 22.08.2012 == | ||
+ | |||
+ | ; PCR | ||
+ | : CFP ([[Team:Marburg_SYNMIKRO/Primer#MS16|MS16]] + [[Team:Marburg_SYNMIKRO/Primer#MS17|MS17]]) | ||
+ | : mRFP ([[Team:Marburg_SYNMIKRO/Primer#MS14|MS14]] + [[Team:Marburg_SYNMIKRO/Primer#MS15|MS15]]) | ||
+ | : initial denaturation 98°C - 5min | ||
+ | : denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 35 times | ||
+ | : 72°C - 5min --> 4°C - 8 | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel25.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:mRFP, 2:mRFP, 3:CFP, 4:CFP (PCR 22.08.2012)]] | ||
+ | |||
+ | ; miniprep | ||
+ | : of LacIQ | ||
+ | |||
+ | ; Test restriction | ||
+ | : of LacIQ with EcoRI and PstI | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel26.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:LacIQ, (control digest 22.08.2012)]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel27.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:GroES, 5-12: GFT-Terminator, (control digest 21.08.2012)]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel28.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-12:Enhancer, (control digest 21.08.2012)]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel29.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:Enhancer, 5-12: P-RBS, (control digest 21.08.2012)]] | ||
+ | |||
+ | : test restriction of Enhancer (->21.08.12), gel 2% | ||
+ | : watch picture | ||
+ | : test restriction of Enhancer and P-RBS (->21.08.12), gel 2% | ||
+ | : watch picture | ||
+ | |||
+ | |||
+ | == 23.08.2012 == | ||
+ | |||
+ | ; PCR | ||
+ | : pSB1K3-BBa_J04450 (template DNA) | ||
+ | : pSB1T3-BBa_J04450 (template DNA) | ||
+ | : used primer --> [[Team:Marburg_SYNMIKRO/Primer#MS24|MS24]] + [[Team:Marburg_SYNMIKRO/Primer#MS25|MS25]] | ||
+ | : initial denaturation 98°C - 5min | ||
+ | : denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times | ||
+ | : 72°C - 5min --> 4°C - 8 | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel31.jpg|thumb|800px|center|M:gene ruler mix ladder, 1-2:pSB1K3-BBa_J04450, 3-4: pSB1T3-BBa_J04450, (PCR 23.08.2012)]] | ||
+ | |||
+ | ; Restriction | ||
+ | : GixR (EcoRI/PstI) | ||
+ | |||
+ | ; Ligation | ||
+ | : GixR in pSB1C3 | ||
+ | |||
+ | ; Transformation | ||
+ | : GixR in pSB1C3 | ||
+ | |||
+ | |||
+ | == 24.08.2012 == | ||
+ | |||
+ | ; PCR | ||
+ | : pSB1A3-BBa_J04450 (template DNA) | ||
+ | : pSB1T3-BBa_J04450 (template DNA) | ||
+ | : pSB1C3-BBa_J04450 (template DNA) | ||
+ | : pSB1K3-BBa_J04450 (template DNA) | ||
+ | : initial denaturation 98°C - 5min | ||
+ | : denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 30 times | ||
+ | : 72°C - 10min --> 4°C - 8 | ||
+ | : primer: [[Team:Marburg_SYNMIKRO/Primer#MS22|MS22]] + [[Team:Marburg_SYNMIKRO/Primer#MS23|MS23]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel32.jpg|thumb|800px|center|M:gene ruler mix ladder, 1-2:pSB1A3-BBa_J04450, 3-4: pSB1K3-BBa_J04450, 5-6:pSB1C3-BBa_J04450, 7-8: pSB1T3-BBa_J04450, (PCR 24.08.2012)]] | ||
+ | |||
+ | ; Restriction | ||
+ | : RBS (XbaI/PstI) | ||
+ | : CFP (EcoRI/PstI) | ||
+ | : mRFP (EcoRI/PstI) | ||
+ | |||
+ | ; Ligation | ||
+ | : mRFP and CFP in pSB1C3 | ||
+ | |||
+ | ; Transformation | ||
+ | : mRFP and CFP in pSB1C3 | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | = Week 8 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp8'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate">27.08. - 02.09.2012</div> | ||
+ | </div> | ||
+ | <div class="hiddenifjs" id="klapp8"> | ||
+ | </html> | ||
+ | |||
+ | |||
+ | == 27.08.2012 == | ||
+ | |||
+ | ; Results | ||
+ | : CFP: 1 colony | ||
+ | : CFP (neu): Colonies | ||
+ | : mRFP: colonies | ||
+ | : mRFP (neu): no colonies | ||
+ | |||
+ | ; PCR | ||
+ | : template: backbones (pSB1K3/T3/C3/A3) | ||
+ | : parameters: 98°C 5min, >> 98°C 1min, 70°C 30s, 72°C 2.5min << x30, 72°C 5min, 4°C 8 | ||
+ | : primer: [[Team:Marburg_SYNMIKRO/Primer#MS22|MS22]] + [[Team:Marburg_SYNMIKRO/Primer#MS23|MS23]] | ||
+ | |||
+ | ; colony-PCR | ||
+ | : parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C 8 | ||
+ | : primer: [[Team:Marburg_SYNMIKRO/Primer#MS18|MS18]] + [[Team:Marburg_SYNMIKRO/Primer#MS19|MS19]] | ||
+ | |||
+ | ; Agarose gel of backbone PCR | ||
+ | : no result | ||
+ | |||
+ | ; elution of the backbones of Friday the 24th | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel33.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:gixR, 3-9: LacIQ-RBS-Gin, 10: CFP, (colony PCR 27.08.2012)]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel34.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:CFP, 5-10: mRFP, (colony PCR 27.08.2012)]] | ||
+ | |||
+ | ; Inoculation | ||
+ | : pSB1C3_... | ||
+ | : mRFP clone 20,24 | ||
+ | : CFP clone 14,15,17,18 | ||
+ | : gix clone 1,2 | ||
+ | : 5ml LB, over night, 37°C | ||
+ | |||
+ | == 28.08.2012 == | ||
+ | |||
+ | ; production of competent TOP10 | ||
+ | |||
+ | ; PCR | ||
+ | : Enhancer, Terminator, Gix, P-RBS | ||
+ | : parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~ | ||
+ | : primer: [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel35.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Terminator, 2-3: Enhancer, 4-6: P-RBS, 7-8: GixR, 9-10: Enhancer, (PCR 28.08.2012)]] | ||
+ | |||
+ | ; Transformation | ||
+ | : [http://partsregistry.org/Part:BBa_I0500 pBad/araC] (plate:1, Well:N14) | ||
+ | : pSacB | ||
+ | : over night, 37°C | ||
+ | |||
+ | ; miniprep | ||
+ | : GixR (1,2) | ||
+ | : CFP (14,15,17,18) | ||
+ | : mRFP (20,24) | ||
+ | : alle in pSB1C3 | ||
+ | |||
+ | == 29.08.2012 == | ||
+ | |||
+ | ; Results | ||
+ | : Transformation: no colonies | ||
+ | |||
+ | ; PCR | ||
+ | : Terminator, Gix, Enhancer, P-RBS | ||
+ | : diluted templates: Terminator, P-RBS, Gix 1 --> 1:10 | ||
+ | : Enhancer, Gix 2 --> 1:20 | ||
+ | : parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~ | ||
+ | : primer: [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel36.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Terminator, 2-3: Enhancer, 4-6: P-RBS, 7-8: GixR, 9: Enhancer control, 10: P-RBS control, 11: GixR control, (PCR 29.08.2012)]] | ||
+ | |||
+ | ; 3AA | ||
+ | : CFP, mRFP with Terminator | ||
+ | : 2µl DNA | ||
+ | : 1µl SpeI | ||
+ | : 2µl Tango | ||
+ | : 15µl dH2O | ||
+ | : --> 1h at 37°C | ||
+ | : +1µl EcoRI | ||
+ | : +2,5µl Tange | ||
+ | : --> 1h at 37°C | ||
+ | : --> inactivation for 20min at 80°C | ||
+ | |||
+ | ; Ligation | ||
+ | : 3AA products in pSB1T3 | ||
+ | : 18.5µl H2O | ||
+ | : 2.5 µl Buffer | ||
+ | : 1µl Insert CFP or mRFP (EcoRI + SpeI) | ||
+ | : 1µl Insert Terminator (BBa_B0010) (XbaI + PstI) | ||
+ | : 1µl pSB1T3 (EcoRI + PstI) | ||
+ | : 1µl Ligase | ||
+ | : --> 1h at RT | ||
+ | |||
+ | ; Transformation | ||
+ | : pBad --> TOP10 + pSB2K3 | ||
+ | : CFP-T, mRFP-T --> TOP10 + pSB1T3 | ||
+ | |||
+ | ; Sequencing | ||
+ | : CFP_fwd | ||
+ | : CFP_rev | ||
+ | : mRFP_fwd | ||
+ | : mRFP_rev | ||
+ | : P-RBS_fwd | ||
+ | : GixR_fwd | ||
+ | : GixR_fwd | ||
+ | : Enhancer_fwd | ||
+ | |||
+ | == 30.08.2012 == | ||
+ | |||
+ | |||
+ | ; Results | ||
+ | : Transformation: no colonies --> testing Trafo with BBa_J04450 A/K/T/C | ||
+ | : 2 colonies at pBad-Trafo from 28.08.2012 --> liquid culture for miniprep | ||
+ | : 1 colony at pSacB-Trafo from 28.08.2012 --> tested on succrose-sensitivity | ||
+ | |||
+ | |||
+ | == 31.08.2012 == | ||
+ | |||
+ | |||
+ | ; PCR | ||
+ | : SacB | ||
+ | : Parameters No.1: 95°C 5', >>95°C 30", 60°C 30", 72°C 1'<< x30, 72°C 5', 4°C | ||
+ | : Parameters No.2: 95°C 5', >>95°C 30", 72°C 1.5'<< x30, 72°C 5', 4°C | ||
+ | : Primer [[Team:Marburg_SYNMIKRO/Primer#MS26|MS26]] + [[Team:Marburg_SYNMIKRO/Primer#MS27|MS27]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel37.jpg|thumb|800px|center|M:1kb gene ruler plus, 1,3,5:SacB, 4,5: none, (PCR 31.08.2012)]] | ||
+ | |||
+ | ; Ligation | ||
+ | : 3AA (CFP-T, mRFP-T) | ||
+ | : watch 29.08. | ||
+ | : used vector: pSB1K3/T3 | ||
+ | |||
+ | ; Transformation | ||
+ | : of ligation in TOP10 | ||
+ | |||
+ | ; miniprep | ||
+ | : pBad/araC K.1+2 | ||
+ | |||
+ | ; restriction | ||
+ | : GixR rehybridization | ||
+ | : cut with EcoRI and PstI | ||
+ | |||
+ | ; ligation | ||
+ | : 1µl GixRhyb E+P | ||
+ | : 1µl pSB1C3 E+P | ||
+ | : 1µl Th-Pol | ||
+ | : 2µl Buffer | ||
+ | : 15µl dH2O | ||
+ | |||
+ | ; Transformation | ||
+ | : ligation + TOP10 --> LB(cam) | ||
+ | |||
+ | ; Results of test-trafo | ||
+ | : Amp, Km, Cm OK --> Km with fewer colonies | ||
+ | : Tet --> hardly colonies | ||
+ | : --> comp.TOP10 obviously all right, exclusion of Tet in future work | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | = Week 9 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp9'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate">03.09. - 09.09.2012</div> | ||
+ | </div> | ||
+ | <div class="hiddenifjs" id="klapp9"> | ||
+ | </html> | ||
+ | |||
+ | |||
+ | == 03.09.2012 == | ||
+ | |||
+ | |||
+ | ; Results | ||
+ | : Transformation | ||
+ | : GixR in C3 --> no colonies | ||
+ | : CFP-T/mRFP-T in C3(31.8.) --> no colonies | ||
+ | : CFP-T/mRFP-T in T3(31.8.) --> many but small colonies | ||
+ | : CFP-T/mRFP-T in T3(29.8.) --> less colonies | ||
+ | |||
+ | |||
+ | ; colony-PCR | ||
+ | : of colonies (CFP-T, mRFP-T) | ||
+ | : parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS18|MS18]] + [[Team:Marburg_SYNMIKRO/Primer#MS19|MS19]] | ||
+ | |||
+ | ; Agarose gel | ||
+ | : 6x colony pT3-CFP-T from 29./31.8. | ||
+ | : 6x colony pT3-mRFP-T from 29./31.8. | ||
+ | : pC3-CFP (14) | ||
+ | : pC3.mRFP (24) | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel38.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-6:CFP-Terminator, 7:CFP control, 8-13:mRFP-Terminator 14: mRFP control, 15-16: none, (colony-PCR 03.09.2012)]] | ||
+ | |||
+ | == 04.09.2012 == | ||
+ | ; Results | ||
+ | : Transformation | ||
+ | : CFP-T/mRFP-T in T3 | ||
+ | : 2 µl -> 5-6 colonies | ||
+ | : 30 µl -> 1-2 colonies | ||
+ | : Gix R in C3 (3.9) --> 4 colonies | ||
+ | |||
+ | ; colony-PCR | ||
+ | : GixR | ||
+ | : CFP-T | ||
+ | : mRFP-T | ||
+ | : parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞ | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | |||
+ | ; gel extraction | ||
+ | : P108 (E/S) | ||
+ | : RBS (X/P) | ||
+ | |||
+ | ; ligation | ||
+ | : SacB1 + pSB1C3 | ||
+ | : P-RBS + pSB1C3 | ||
+ | |||
+ | ; Transformation | ||
+ | : ligation + TOP10 --> LB(cmp) | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel40.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:pBAD/araC, (mini-prep 31.08.2012)]] | ||
+ | |||
+ | |||
+ | ; Inoculation | ||
+ | : pBad/araC | ||
+ | : 5ml LB, over night, 37°C | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel41.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:GixR pSB1C3, 5-9:CFP-Terminator, 10:CFP control, (colony-PCR 04.09.2012)]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel42.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-9:mRFP-Terminator, 10:mRFP control, (colony-PCR 04.09.2012)]] | ||
+ | |||
+ | |||
+ | == 05.09.2012 == | ||
+ | ; Results | ||
+ | : Transformation | ||
+ | : SacB in C3 | ||
+ | : P-RBS in C3 | ||
+ | |||
+ | ; colony PCR | ||
+ | : SacB in C3 | ||
+ | : P-RBS in C3 | ||
+ | : parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞ | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | |||
+ | ; miniprep | ||
+ | : Gix in C3 | ||
+ | : pBad | ||
+ | |||
+ | ; Results | ||
+ | : Transformation | ||
+ | : CFP-T (03.09) --> 10 colonies | ||
+ | |||
+ | ; colony PCR | ||
+ | : P108-RBS, SacB, GixR | ||
+ | : parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞ | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS19|MS19]] + [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel43.jpg|thumb|800px|center|'''no marker''', 1:none, 2-7:SacB, 8-11:P108-RBS, (colony-PCR 05.09.2012)]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel44.jpg|thumb|800px|center|'''no marker''', 1-5:P108-RBS, 6:none, 7-9:GixR, 10:Scar, 11-12: pBad, 13-15: pGixR, (colony-PCR 05.09.2012)]] | ||
+ | |||
+ | |||
+ | ; restriction | ||
+ | : pSB1A3 (E/P) | ||
+ | : pSB1K3 (E/P) | ||
+ | : pSB1C3 (E/P) | ||
+ | : pSB1T3 (E/P) | ||
+ | : GFP-T K3 (X/P) | ||
+ | : GFP K3 (X/P) | ||
+ | : CFP C3 (X/P) | ||
+ | : mRFP C3 (X/P) | ||
+ | : AmiC A (E/S) | ||
+ | : GroES A (E/S) | ||
+ | : Terminator (X/P) | ||
+ | |||
+ | |||
+ | ; Inoculation | ||
+ | : pRBS | ||
+ | : SacB | ||
+ | : mRFP-Terminator | ||
+ | : 5ml LB, over night, 37°C | ||
+ | |||
+ | == 06.09.2012 == | ||
+ | |||
+ | ; miniprep | ||
+ | : pRBS | ||
+ | : SacB | ||
+ | : mRFP-Terminator | ||
+ | |||
+ | ; ligation | ||
+ | : AmiC (E/S) + GFP (X/P) + pSB1T3 (E/P) | ||
+ | : AmiC (E/S) + RFP (X/P) + pSB1T3 (E/P) | ||
+ | : AmiC (E/S) + GCFP (X/P) + pSB1T3 (E/P) | ||
+ | : AmiC (E/S) + Terminator (X+P + pSB1T3 (E/P) | ||
+ | : GroES (E/S) + GFP (X/P) + pSB1T3 (E/P) | ||
+ | : GroES (E/S) + RFP (X/P) + pSB1T3 (E/P) | ||
+ | : GroES (E/S) + CFP (X/P) + pSB1T3 (E/P) | ||
+ | : GroES (E/S) + Terminator (X/P) + pSB1T3 (E/P) | ||
+ | |||
+ | ; PCR of miniprep | ||
+ | : SacB in pSB1C3 | ||
+ | : p-RBS in pSB1C3 | ||
+ | : terminator in pSB1T3 | ||
+ | : parameters: 98°C 5min, >> 98°C 30 sec, 72°C 1,5min << x30, 72°C 5min, 4°C ∞ | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel45.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Scar, 2-5:P-RBS, 6-9:SacB, 10-16: mRFP, 17: control, (PCR of miniprep (06.09.2012)]] | ||
+ | |||
+ | |||
+ | ; transformation | ||
+ | : RBS-Gin T3 | ||
+ | : amiC A3 | ||
+ | : groES A3 | ||
+ | : groES-Terminator T3 | ||
+ | : groES-GFP T3 | ||
+ | : groES-CFP T3 | ||
+ | : groES-mRFP T3 | ||
+ | : amiC-Terminator T3 | ||
+ | : amiC-GFP T3 | ||
+ | : amiC-CFP T3 | ||
+ | : amiC-mRFP T3 | ||
+ | |||
+ | == 07.09.2012 == | ||
+ | |||
+ | ; restriction (6.9.) | ||
+ | |||
+ | ; ligation | ||
+ | : AmiC in pSB1K3 | ||
+ | : GroES in pSB1K3 | ||
+ | : mRFP in pSB1K3 | ||
+ | : Terminator in pSB1K3 | ||
+ | : CFP in pSB1K3 | ||
+ | : GFP in pSB1K3 | ||
+ | : GFP+Terminator in C3 | ||
+ | : Enhancer in K3 | ||
+ | |||
+ | ; transformation | ||
+ | : Ligation | ||
+ | : AmiC | ||
+ | : GroES | ||
+ | : RBS-Gin | ||
+ | |||
+ | ; miniprep | ||
+ | : CFP-Terminator | ||
+ | |||
+ | ; Sequencing | ||
+ | : mRFP T3 | ||
+ | : P-RBS K12 | ||
+ | : P-RBS K15 | ||
+ | : SacB K6 | ||
+ | : SacB K6 | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | = Week 10 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp10'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate"> 10.09. - 16.09.2012</div> | ||
+ | </div> | ||
+ | <div class="hiddenifjs" id="klapp10"> | ||
+ | </html> | ||
+ | |||
+ | ==10.09.2012== | ||
+ | ; Restriction | ||
+ | : Gin-RBS (01.08) | ||
+ | |||
+ | ; Ligation | ||
+ | : Gin-RBS in pSB1C3 | ||
+ | |||
+ | ; Transformation | ||
+ | : Gin-RBS in pSB1C3 | ||
+ | |||
+ | ; Hybridisation | ||
+ | : GixR | ||
+ | : 98 °C 20 min | ||
+ | : minus 1 °C to 4 °C in 2 min | ||
+ | |||
+ | ; Ligation and Transformation | ||
+ | : GixR (X+S) + pSB1K3 (X+S) | ||
+ | |||
+ | ; Inoculation (Tetracycline) | ||
+ | : GroES-mRFP | ||
+ | : GroES-GFP | ||
+ | : GroES-CFP | ||
+ | : amiC-mRFP | ||
+ | : amiC-GFP | ||
+ | : amiC-CFP | ||
+ | : 5ml LB, over night, 37°C | ||
+ | |||
+ | ; Inoculation (Kanamycine) | ||
+ | : GroES-GFP | ||
+ | : amiC-mRFP | ||
+ | : amiC-GFP | ||
+ | : GroES-Terminator | ||
+ | : Enhancer | ||
+ | : 5ml LB, over night, 37°C | ||
+ | |||
+ | ; Inoculation (Chloramphenicol) | ||
+ | : GFP-Terminator | ||
+ | : 5ml LB, over night, 37°C | ||
+ | |||
+ | ; Inoculation (Ampicillin) | ||
+ | : Terminator | ||
+ | : 5ml LB, over night, 37°C | ||
+ | |||
+ | == 11.09.2012 == | ||
+ | ;microscopy-screening | ||
+ | :GroES-GFP ⇒; no fluorescence | ||
+ | :GroES-CFP ⇒; no fluorescence | ||
+ | :AmiC-mRFP ⇒; no fluorescence | ||
+ | :AmiC-GFP ⇒; no fluorescence | ||
+ | |||
+ | ; colony-PCR | ||
+ | : GixR | ||
+ | : RBS-Gin | ||
+ | : Terminator | ||
+ | : parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | |||
+ | ;3A-Assembly (digestion, ligation, transformation) | ||
+ | :GroES (E/S) + Terminator (X/P) + pSB1K3 (P/E) | ||
+ | :AmiC (E/S) + Terminator (X/P) + pSB1K3 (P/E) | ||
+ | |||
+ | ;plasmid-prep and control-digestion (E/P) | ||
+ | :GroES-GFP | ||
+ | :GroES-CFP | ||
+ | :AmiC-mRFP | ||
+ | :AmiC-GFP | ||
+ | :GroES-Terminator | ||
+ | :Enhancer | ||
+ | :GFP-Terminator | ||
+ | :Terminator | ||
+ | |||
+ | == 12.09.2012 == | ||
+ | ; Results | ||
+ | : Transformation | ||
+ | : - P-RBS --> colonies | ||
+ | : - pB10 --> no colonies | ||
+ | : - RBS (B0030) --> colonies | ||
+ | : - Gro-ES/ AmiC-Terminator --> no colonies | ||
+ | |||
+ | ; colony-PCR | ||
+ | : Gix (2. try) | ||
+ | : Gin-RBS (2. try) | ||
+ | : RBS (B0030) | ||
+ | : P-RBS (108) | ||
+ | : parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | |||
+ | ; Agarose gel | ||
+ | : Gin-RBS (5x) | ||
+ | : Gin (1x) | ||
+ | : Gix (5x) | ||
+ | : RBS (6x) | ||
+ | : P-RBS (6x) | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel46.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Scar, 2-6:RBS-Gin, 7:Gin control 8-12: gixR, 13: control, (colony-PCR 12.09.2012)]] | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel47.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Scar, 2-6:RBS BBa_B0030, 7-12:P-RBS, 13: control, (colony-PCR 12.09.2012)]] | ||
+ | |||
+ | |||
+ | ; Restriction (miniprep 11.09.12) | ||
+ | : gr-GFP | ||
+ | : gr-CFP | ||
+ | : gr-T K3 | ||
+ | : enhancer K3 | ||
+ | : am-RFP-K3 | ||
+ | : am-GFP-K3 | ||
+ | : GFP-T-C3 | ||
+ | : terminator A2 | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel48.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-3:GroES-GFP, 4:GroES-GFP control, 5:GroES-CFP, 6:GroES-CFP control, 7-10: GroES-Terminator, 11: GroES-Terminator control, 12-15: Enhancer, 16: Enhancer control, (control digest 12.09.2012)]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel49.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:AmiC-mRFP, 3:AmiC-mRFP control, 4-5:AmiC-GFP, 6:AmiC-GFP control, 7-11: GFP-Terminator, 12: GFP-Terminator control, 13-15: Terminator, 16: Terminator control, (control digest 12.09.2012)]] | ||
+ | |||
+ | ; Transformation | ||
+ | : groES-T | ||
+ | ::2 Transformations: | ||
+ | ::: - One with a 1:10 dilution of Terminator from ligation, | ||
+ | ::: - another one with a 1:100 dilution of Terminator from ligation | ||
+ | : amiC-T | ||
+ | ::2 Transformations: | ||
+ | ::: - One with a 1:10 dilution of Terminator from ligation, | ||
+ | ::: - another one with a 1:100 dilution of Terminator from ligation | ||
+ | |||
+ | ; PCR | ||
+ | : Gin-RBS | ||
+ | ::3 reactions: | ||
+ | ::: -One using High Fidelity (HF) Reaction Buffer, | ||
+ | ::: -one using GC-Buffer | ||
+ | ::: -and another one using self-made reaction buffer | ||
+ | : Primer used : [[Team:Marburg_SYNMIKRO/Primer#MS12|MS12]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]] | ||
+ | : Template DNA : ''E. coli'' C600 chromosomal DNA | ||
+ | |||
+ | == 13.09.2012 == | ||
+ | ; Results | ||
+ | : Transformation | ||
+ | :: - pBAD (11.09.12) --> no colonies | ||
+ | :: - AmiC-T (11.09.12) --> 2 x 2 colonies | ||
+ | :: - GroES-T (11.12.09)--> 2 colonies | ||
+ | |||
+ | ; colony-PCR | ||
+ | : AmiC-T & GroES-T (transformation 11.09.12) | ||
+ | : parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel50.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:RBS-Gin HF, 2:RBS-Gin GC, 3:RBS-Gin SM (PCR 13.09.2012)]] | ||
+ | |||
+ | |||
+ | ; Gel extraction | ||
+ | : RBS-Gin (HF) --> 51 ng/µl | ||
+ | : RBS-Gin (GC) --> 69 ng/µl | ||
+ | : RBS-Gin (SM) --> 56 ng/µl | ||
+ | |||
+ | ; Ligation | ||
+ | : GroES-T & AmiC-T in pSB1K3 | ||
+ | |||
+ | ; Restriction (preparative digest) | ||
+ | : GroES (A module) | ||
+ | :: with SpeI | ||
+ | : AmiC (A module) | ||
+ | :: with SpeI | ||
+ | : mRFP-C3 (B module) | ||
+ | :: with EcoRI and XbaI | ||
+ | : CFP-C3 (B module) | ||
+ | :: with EcoRI and XbaI | ||
+ | : Gin-RBS | ||
+ | :: HF-PCR-Product, GC-PCR-product and SM-PCR-product | ||
+ | ::: with EcoRI and PstI | ||
+ | |||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel51.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:AmiC-Terminator, 5-6:GroES-Terminator, 7:Scar, 8: control, (colony-PCR 13.09.2012)]] | ||
+ | |||
+ | |||
+ | == 14.09.2012 == | ||
+ | |||
+ | ; Results | ||
+ | : Transformation | ||
+ | :: AmiC-T: no colonies | ||
+ | :: GroES-T: no colonies | ||
+ | |||
+ | ; Restriction | ||
+ | : 2nd Digestion of A-modules (see digest from yesterday) additionally with EcoRI | ||
+ | |||
+ | ; Ligation | ||
+ | : Gin-RBS with pSB1C3 | ||
+ | :: 3 reactions: | ||
+ | ::: one with RBS-Gin HF-PCR-Product | ||
+ | ::: one with RBS-Gin GC-PCR-Product | ||
+ | ::: one with RBS-Gin SM-PCR-Product | ||
+ | |||
+ | ; Inoculation | ||
+ | : Gix-K3 (clone 3 and 4) | ||
+ | : P-RBS-C3 (clone 1 and 3) | ||
+ | : RBS (BBa_B0030) [clone 1 and 3] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel52.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:AmiC, 2:GroES, 3:none, 4:GFP, 5:CFP, 6:mRFP,(digest 13.-14.09.2012)]] | ||
+ | |||
+ | ; Gel extraction | ||
+ | : GFP | ||
+ | : mRFP | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel53.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Terminator, (digest 14.09.2012)]] | ||
+ | |||
+ | ; Gel extraction | ||
+ | : Terminator clone 48 | ||
+ | |||
+ | ; Transformation | ||
+ | : RBS-Gin(HF)-pSB1C3 | ||
+ | : RBS-Gin(GC)-pSB1C3 | ||
+ | : RBS-Gin(SM)-pSB1C3 | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div class="conboxb"> | ||
+ | </html> | ||
+ | = Week 11 = | ||
+ | <html> | ||
+ | <div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp11'); togglearrow(this); return false"></div> | ||
+ | <div class="smalldate"> 17.09. - 23.09.2012</div> | ||
+ | </div> | ||
+ | <div class="hiddenifjs" id="klapp11"> | ||
+ | </html> | ||
+ | |||
+ | == 17.09.2012 == | ||
+ | |||
+ | ; Restriction | ||
+ | : AmiC | ||
+ | : GroES | ||
+ | : CFP | ||
+ | : SacB | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel54.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:AmiC control, 2:AmiC, 3:GroES control, 4:GroES, 5:CFP control, 6:CFP, 7:SacB control, 8:SacB (digest 17.09.2012)]] | ||
+ | |||
+ | ; Restriction | ||
+ | : CFP-C3 | ||
+ | : SacB (2nd try) | ||
+ | |||
+ | ; Resuspend | ||
+ | : BBa_B001J | ||
+ | |||
+ | ; Restreak | ||
+ | : RBS-Gin (HF) | ||
+ | : RBS-Gin (GC) | ||
+ | : RBS-Gin (SM) | ||
+ | |||
+ | ; DNA isolation | ||
+ | : CFP-C3 (Miniprep28.08.12)--> 47,3 ng/µl | ||
+ | |||
+ | |||
+ | ; Miniprep | ||
+ | : GixR pSB1K3 --> 50,5 ng/µl, 144,7 ng/µl | ||
+ | : RBS(new)B0030 pSB1A2 --> 726,2 ng/µl, 242,6 ng/µl | ||
+ | : P108-RBS pSB1C3 --> 200,5 ng/µl, 177,8 ng/µl | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel55.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:GixR, 3-4:RBS BBa_B0030, 3-4:P108-RBS, (mini prep 17.09.2012)]] | ||
+ | |||
+ | ; Transformation | ||
+ | : pSB2K3-pBAD & pSB1-A2-TT | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : CFP-C3 (restriction 2nd try) | ||
+ | : SacB-Cs (restriction 2nd try) | ||
+ | : [[File:Syn_MR_Gel56.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:CFP control, 2:CFP, 3:SacB, (digest 17.09.2012)]] | ||
+ | |||
+ | == 18.09.2012 == | ||
+ | |||
+ | ; Results | ||
+ | ; Transformation | ||
+ | : 2x Terminator --> colonies | ||
+ | : pBAD --> no colonies | ||
+ | |||
+ | ; Colony PCR | ||
+ | : RBS-Gin | ||
+ | : 2x Terminator | ||
+ | : parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel57.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:RBS-Gin HF, 3:RBS-Gin GC, 4:RBS-Gin SM, 5: Scar, 7-10:2xTerminator, 11:control, (colony-pcr 18.09.2012)]] | ||
+ | |||
+ | ; Sequencing | ||
+ | : SacB | ||
+ | |||
+ | ; Gel extraction | ||
+ | : CFP-C3 E+X | ||
+ | |||
+ | ; Ligation | ||
+ | : am-G: amiC E+S & GFP-A3 E+X | ||
+ | : am-C: amiC E+S & CFP-C3 E+X | ||
+ | : am-R: amiC E+S & mRFP-C3 E+X | ||
+ | : gro-G: groES E+S & GFP-A3 E+X | ||
+ | : gro-C: groES E+S & CFP-C3 E+X | ||
+ | : gro-R: groES E+S & mRFP-C3 E+X | ||
+ | |||
+ | ; Transformation | ||
+ | : am-G | ||
+ | : am-C | ||
+ | : am-R | ||
+ | : gr-G | ||
+ | : gr-C | ||
+ | : gr-R | ||
+ | : pBAD | ||
+ | |||
+ | |||
+ | == 19.09.2012 == | ||
+ | |||
+ | ; Results | ||
+ | ; Transformation | ||
+ | : am-G --> colonies | ||
+ | : am-C --> colonies | ||
+ | : am-R --> colonies | ||
+ | : gr-G --> colonies | ||
+ | : gr-C --> colonies | ||
+ | : gr-R --> colonies | ||
+ | : pBAD --> no colonies | ||
+ | |||
+ | ; Colony PCR | ||
+ | : RBS-Gin (from 17.09.12) | ||
+ | : 2x Terminator B0017 (from 17.09.12) | ||
+ | : parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C | ||
+ | : primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]] | ||
+ | : --> Amplification failed | ||
+ | |||
+ | ; Transformation | ||
+ | : pBAD | ||
+ | |||
+ | ; Microscopy-screening | ||
+ | : AmiC-GFP --> fluorescence | ||
+ | : GroES-GFP --> fluorescence | ||
+ | : AmiC-CFP --> fluorescence | ||
+ | : GroES-CFP --> fluorescence | ||
+ | : AmiC-mRFP --> fluorescence | ||
+ | : GroES-mRFP --> fluorescence | ||
+ | |||
+ | ; Inoculation | ||
+ | : RBS-Gin | ||
+ | : TT (B0017) | ||
+ | : AmiC-GFP | ||
+ | : GroES-GFP | ||
+ | : AmiC-CFP | ||
+ | : GroES-CFP | ||
+ | : AmiC-mRFP | ||
+ | : GroES-mRFP | ||
+ | |||
+ | |||
+ | == 20.09.2012 == | ||
+ | |||
+ | ; Miniprep | ||
+ | : RBS-Gin | ||
+ | : TT (B0017) | ||
+ | : AmiC-GFP | ||
+ | : GroES-GFP | ||
+ | : AmiC-CFP | ||
+ | : GroES-CFP | ||
+ | : AmiC-mRFP | ||
+ | : GroES-mRFP | ||
+ | |||
+ | ; Restriction (E/P) | ||
+ | : TT(B0017) | ||
+ | : RBS-Gin | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel58.jpg|thumb|800px|center|M:1kb gene ruler plus, 1,3,5,7:2xTerminator, 2,4,6,8:2xTerminator control, 9,11,13,15:RBS-Gin, 10,12,14,16:RBS-Gin control, (control digest 20.09.2012)]] | ||
+ | |||
+ | ; Restriction | ||
+ | : SacB ((XbaI + PstI) & (EcoRI + XbaI) | ||
+ | : P-RBS (EcoRI + SpeI) & (SpeI + PstI) | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel59.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:P-RBS control, 2: P-RBS (E/S), 3: P-RBS (S/P), 4: SacB control, 5: SacB (X/P), 6:SacB (E/X), (digest 20.09.2012)]] | ||
+ | |||
+ | ; Inoculation | ||
+ | : GroES-GFP | ||
+ | : GroES-CFP | ||
+ | : GroES-mRFP | ||
+ | |||
+ | |||
+ | == 21.09.2012 == | ||
+ | |||
+ | |||
+ | ; Restriction | ||
+ | : GFP-fusion (EcoRI + SpeI) | ||
+ | : TT BBa_0017 (EcoRI + XbaI) | ||
+ | |||
+ | ; 1% Agarose gel | ||
+ | : [[File:Syn_MR_Gel60.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:GFP-Fusion, 2:TT BBa_B0017, 3:SacB, (digest 21.09.2012)]] | ||
+ | |||
+ | ; Gel extraction | ||
+ | : GFP-Fusion | ||
+ | : pSB1A2 (backbone GFP-Fusion) | ||
+ | : TT | ||
+ | : SacB | ||
+ | |||
+ | ; Ligation | ||
+ | : GFP bb Enhancer | ||
+ | : GFP TT | ||
+ | : GroES TT | ||
+ | |||
+ | ; Transformation | ||
+ | : GFP bb Enhancer | ||
+ | : GFP TT | ||
+ | : GroES TT | ||
+ | : Promotor-RBS (17.09.12) | ||
+ | |||
+ | ; Sequencing | ||
+ | : RBS-Gin | ||
+ | |||
+ | ; Microscopy-screening | ||
+ | : AmiC-GFP --> Channel: DIC, Green, Red, Blue | ||
+ | : GroES-GFP --> Channel: DIC, Green, Red, Blue | ||
+ | : AmiC-CFP --> Channel: DIC, Green, Red, Blue | ||
+ | : GroES-CFP --> Channel: DIC, Green, Red, Blue | ||
+ | : AmiC-mRFP --> Channel: DIC, Red | ||
+ | : GroES-mRFP --> Channel: DIC, Green, Red, Blue | ||
+ | |||
+ | == 22.09.2012 == | ||
+ | |||
+ | ; Inoculation | ||
+ | : GroES-TT pSB1A2 | ||
+ | : GFP-TT pSB1A2 | ||
+ | : Enhancer | ||
+ | : P108-RBS pSB1C3 | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | </script> | ||
+ | <script type="text/javascript"> | ||
+ | function ReplaceContentInContainer(matchClass) | ||
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+ | } | ||
+ | </script> | ||
+ | </html> | ||
- | + | {{:Team:Marburg_SYNMIKRO:Template:Footer}} |
Latest revision as of 01:44, 27 September 2012
Contents |
Week 1
[all]
09.07. - 15.07.2012
09.07.2012
- Inoculation
- E. coli strain C600 Mucts62 in dYT-medium
10.07.2012
- Isolation of chromosomal DNA
- E. coli strain C600 Mucts62 (lysogenic for temperature sensitive phage Mu cts62)
- Inoculation
- E. coli strain DH5α and TOP10 in dYT-medium
11.07.2012
- Preparation of chemically competent E. coli cells
- TOP10
12.07.2012
- Transformation
- GFP, GFP_fusion, CFP, mRFP, RBS, P108, P100, Plac with E.coli TOP10
Name | Biobrick | Plate | Well | Backbone | Resistance |
---|---|---|---|---|---|
GFP | [http://partsregistry.org/Part:BBa_E0040 BBa_E0040] | 1 | 14K | pSB1A2 | Ampicillin |
GFP_fusion | [http://partsregistry.org/Part:BBa_K125500 BBa_K125500] | 3 | 2P | pSB1A2 | Ampicillin |
CFP | [http://partsregistry.org/Part:BBa_E0020 BBa_E0020] | 1 | 6A | pSB1A2 | Ampicillin |
mRFP | [http://partsregistry.org/Part:BBa_E1010 BBa_E1010] | 1 | 18F | pSB2K3 | Kanamycin |
RBS | [http://partsregistry.org/Part:BBa_J61100 BBa_J61100] | 1 | 5J | pSB1A2 | Ampicillin |
P108 | [http://partsregistry.org/Part:BBa_J23108 BBa_J23108] | 2 | 2E | BBa_J61002 | Ampicillin |
P100 | [http://partsregistry.org/Part:BBa_J23100 BBa_J23100] | 1 | 18C | BBa_J61002 | Ampicillin |
PLac | [http://partsregistry.org/Part:BBa_I14032 BBa_I14032] | 2 | 11P | pSB2K3 | Kanamycin |
Week 2
16.07. - 22.07.2012
16.07.2012
- PCR
- AmiC (primer: MS1 + MS2)
- HU (primer: MS3 + MS4)
- GroES (primer: MS5 + MS6)
- GixR (primer: MS7 + MS8)
- Enhancer (primer: MS9 + MS10)
- ⇒ template-DNA: chromosomal DNA, denaturation: 30 sec at 95°C; annealing: 30 sec at 60°C; elongation: 30 sec at 72°C; 35 cycles
- Inoculation
- GFP
- GFP_fusion
- CFP
- mRFP
- RBS
- P108
- LacIQ
- P100
17.07.2012
- Plasmid preparation
- GFP
- GFP_fusion
- CFP
- mRFP
- RBS
- P108
- LacIq
- P100
18.07.2012
- 1% Agarose gel
- DNA Gel extraction
- Enhancer
- GroES
- HU
- GixR Hybridization
19.07.2012
- Fill-in reaction (primer extension)
- Enhancer
- Restriction
- Enhancer (EcoRI/PstI)
- ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 85 °C, elongation: 36 sec; 72 °C, 35 cycles, 1) 2 µl DMSO; 1 µl chrom. DNA | 2) 2 µl DMSO; 5 µl chrom. DNA | 3) without DMSO; 1 µl chrom. DNA | 4) without DMSO; 5 µl chrom. DNA | 5) 2 µl DMSO; without chrom. DNA
- Test restriction digest
- 7) CFP (EcoRI/PstI)
- 8) GFP (EcoRI/PstI)
- 9) GFP-fusion (EcoRI/PstI)
- 10) mRFP (EcoRI/PstI)
- ⇒ all negative
- 1% Agarose gel
- Control digest
- CFP (EcoRI/PstI)
- GFP (EcoRI/PstI)
- GFP-fusion (EcoRI/PstI)
- mRFP (EcoRI/PstI)
- ⇒ restriction over night
20.07.2012
- 1% Agarose gel from the control digest
Week 3
23.07. - 29.07.2012
Week 4
30.07. - 05.08.2012
30.07.2012
- PCR
- CFP (primer: MS16 + MS17) ⇒ template-DNA: [http://partsregistry.org/Part:BBa_E0020 BBa_E0020]
- ⇒ positive
- mRFP (primer: MS14 + MS15) ⇒ template-DNA: [http://partsregistry.org/Part:BBa_E1010 BBa_E1010]
- ⇒ positive
- Gin (primer: MS11 + MS13) ⇒ template-DNA: chromosomal DNA (that was wrong)
- RBS-Gin (primer: MS13 + MS12) ⇒ template-DNA: chromosomal DNA (that was wrong)
- ⇒ annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
- Transformation
- all vector-backbone-ligations
- Enhancer (K3)
- GixR (A3)
31.07.2012
- Result
- transformation
- pSB1A3: 3 colonies
- pSB1K3: 60 colonies
- pSB1C3: 9 colonies
- pSB1T3: 0 colonies
- Enhancer (K3): 0 colonies
- GixR (A3): 0 colonies
- 1% Agarose gel
- 1% Agarose gel
- 1% Agarose gel
- Resuspension of a biobrick
- [http://partsregistry.org/Part:BBa_B0010 BBa_B0010](Terminator)
- DNA Gel extraction
- CFP
- mRFP
- PCR
- AmiC (primer: MS1 + MS2)
- RBS-Gin (primer: MS12 + MS13)
- Gin (primer: MS11 + MS13)
- ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 40 sec; 72 °C, 35 cycles
- Restriction
- pSB1A3 (EcoRI/PstI)
- CFP (EcoRI/PstI)
- mRFP (EcoRI/PstI)
- Transformation
- pSB1A3
- pSB1C3
- pSB1T3
- Terminator
- GixR
- Enhancer
- Inoculation
- pSB1A3
- pSB1C3
- pSB1K3
- Ligation
- pSB1A3 + CFP
- pSB1A3 + mRFP
- GixR Hybridization
- DNA Gel extraction
- AmiC
- Ligation
- AmiC + pSV2 (suicide vector)
01.08.2012
- Result
- transformation
- Only BBa_B0010 colonies present
- Plasmid preparation
- pSB1A3 ⇒ only clone 1, 3 Positive
- pSB1C3 ⇒ all negative
- pSB1K3 ⇒ all negative
- Ligation
- GixR hyb. + pJET2_1
- ⇒ over blunt ends
- Restriction
- GixR hyb. (EcoRI/PstI)
- pSB1C3 (EcoRI/PstI)
- 1% Agarose gel
- 1% Agarose gel
- DNA Gel extraction
- RBS-Gin
- Gin
- AmiC
- Test restriction
- pSB1A3 (EcoRI)
- Restriction
- RBS-Gin (EcoRI/PstI)
- Gin (EcoRI/PstI)
- AmiC (EcoRI/PstI)
- pSB1C3 (EcoRI/PstI)
- pSB1K3 (EcoRI/PstI)
- pSB1T3 (EcoRI/PstI)
- Ligation
- cyclisation of pSB1T3
- Fill-in reaction (primer extension)
- Enhancer
- Ligation
- Enhancer and GixR in pSV2
- Enhancer in pSB1K3
- GixR in pSB1C3
- 1% Agarose gel
- pSB1A3 c2
- pSB1C3 c4
- pSB1K3 c3
- Transformation
- pSB1A3 religation
- CFP in A3
- mRFP in A3
- AmiC in pJET
- GixR in pJET
- Enhancer in pJET
- GixR in C3
- Enhancer in K3
- Ligation
- AmiC + pSB1T3
- RBS-Gin + pSB1K3
- Gin + pSB1K3
02.08.2012
- Resuspension of a biobrick
- pSB3C5
- ⇒ for ccdB
- Plasmid preparation
- BBa_B0010 (terminator)
- Test restriction
- Terminator c1,2,3,4 (EcoRI/PstI)
- 2% Agarose gel
- Transformation
- pSB1T3 recycled
- AmiC in T3
- Gin-RBS in K3
- Gin in K3
- pSB3C5 linear (false)
03.08.2012
- Plasmid preparation
- Enhancer in pJET
Week 5
06.08. - 12.08.2012
06.08.2012
- Resuspension and transformation of a biobrick
- BBa_J04450 in pSB1A3
- BBa_J04450 in pSB1C3
- BBa_J04450 in pSB1K3
- BBa_J04450 in pSB1T3
- Restriction
- GFP-fusion (EcoRI/SpeI)
- Terminator (XbaI/PstI)
- Promoter BBa_J23100 (EcoRI/SpeI)
- Promoter BBa_J23108 (EcoRI/SpeI)
- RBS (XbaI/PstI)
- Ligation
- RBS-Gin in T3
- Gin in T3
- GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3
- Promoter BBa_J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3
- Promoter BBa_J23100 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3
- GixR Hybridization
- 1(3) µl primer MS77 (100 µM) + 1(3) µl primer MS8 (100 µM) + T4-Ligasebuffer
- 5 min 95°C
- over night, room temperature
- -80°C
- Transformation
- GixR in C3
- Gin in T3
- RBS-Gin in T3
- mRFP in A3
- CFP in A3
07.08.2012
- Transformation
- GFP-fusion
- CFP
- J23108-RBS
- J23100-RBS
- Plasmid preparation
- GixR 1,2,3
- Restriction
- GixR hyb. 1,2,3 (EcoRI/PstI)
- pSB1C3 (EcoRI/PstI)
- Ligation
- GixR hyb. 1,2,3 in pJET
- GixR hyb. 1,2,3 in C3
- Transformation
- GixR hyb. 1,2,3 in pJET
- GixR hyb. 1,2,3 in C3
- Test restriction
- pSB3C5 (XbaI)
- pJET (XbaI)
- 1% Agarose gel
- ⇒ positive: pSB3C5 3,4,7,8,; Enhancer-pJET 9-12
08.08.2012
- Result transformation
- GFP-fusion (20 clones)
- J23108-RBS (20 clones)
- J23100-RBS (20 clones)
- GixR hyb. 1,2,3 in pJET (10 clones)
- GixR hyb. 1,2,3 in C3 (10 clones)
- CFP (5 clones)
- Restriktion
- Enhancer in pJET (EcoRI/PstI)
- pSB1C3 (EcoRI/PstI)
- 2% Agarose gel
- DNA Gel extraction
- Enhancer
- pSB1C3
- Ligation
- Enhancer 1,2 in C3
- Transformation
- Enhancer 1,2 in C3
- C3
- mRFP
09.08.2012
- Result transformation
- GFP-fusion ⇒ positive
- J23108-RBS ⇒ positive
- J23100-RBS ⇒ positive
- CFP ⇒ positive
- GixR in C3 c1 ⇒ negative
- GixR in C3 c2 ⇒ negative
- GixR in C3 c3 ⇒ negative
- Plasmid preparation
- GFP-fusion
- J23108-RBS
- J23100-RBS
- CFP
- Test restriction
- GFP-fusion (EcoRI/PstI)
- J23108-RBS (EcoRI)
- J23100-RBS (EcoRI)
- CFP (EcoRI/PstI)
- 1% Agarose gel
- 1% Agarose gel
- 1% Agarose gel
- 1% Agarose gel
- Inoculation
- GixR in C3
- Gin in T3
- Gin-RBS in T3
- pSB1C3
- Enhancer in C3
- mRFP in A3
Week 6
13.08. - 19.08.2012
13.08.12
- PCR
- GroES (primer: MS5 + MS6b)
- HU (primer: MS3 + MS4b)
- ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
- 1% Agarose gel
- Transformation
- LacIQ (BBa_I14032)
14.08.12
- Restriction
- pSB1K3 (EcoRI/PstI)
- Enhancer 3,4 (EcoRI/ PstI)
- GFP-fusion (EcoRI/SpeI)
- Terminator (XbaI/PstI)
- Test restriction
- RBS-Gin in T3 (EcoRI/ PstI)
- Ligation
- GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3 (EcoRI/PstI)
- Promotor J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3 (EcoRI/PstI)
- Enhancer 3,4 (EcoRI/ PstI) + pSB1K3 (EcoRI/PstI)
- Transformation
- GFP-fusion-Terminator in pSB1K3
- Promotor-RBS in pSB1K3
- Enhancer 3 in pSB1K3
- Enhancer 4 in pSB1K3
- GixR in pJET
15.08.2012
- PCR
- GroES (primer: MS5 + MS6b)
- HU (primer: MS3 + MS4b)
- ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C, 35 cycles
- 1% Agarose gel
- DNA Gel extraction
- HU 1,2
- Resuspension of a biobrick
- BBa_J04450 in pSB1A3
- BBa_J04450 in pSB1T3
- BBa_J04450 in pSB1C3
- BBa_J04450 in pSB1K3
- Transformation
- BBa_J04450 in pSB1A3
- BBa_J04450 in pSB1T3
- BBa_J04450 in pSB1C3
- BBa_J04450 in pSB1K3
- Test restriction
- pSB1T3 (EcoRI/PstI and DpnI)
- pSB1K3 (EcoRI/PstI and DpnI)
- pSB1C3 (EcoRI/PstI and DpnI)
- pSB1A3 (EcoRI/PstI and DpnI)
16.08.2012
- PCR
- GroES (primer: MS5 + MS6b)
- ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
- 1% Agarose gel
- Plasmid preparation
- P-RBS
- Test restriction
- P-RBS (EcoRI/PstI)
- 1% Agarose gel
- Restriction
- Enhancer 3,4 (EcoRI/ PstI)
- HU (EcoRI/ PstI)
- AmiC (EcoRI/ PstI)
- Promoter, GFP (EcoRI/SpeI)
- RBS; Terminator (XbaI/PstI)
- Ligation
- HU, AmiC in pSB1A3
- Enhancer 3,4 in pSB1K3
- P-RBS in pSB1K3
- GFP fusion-Terminator in pSB1K3
- DNA Gel extraction
- GroES 1, P-RBS
- Transformation
- AmiC
- HU
- Enhancer 3,4, 3 alt,4 alt
- Inoculation
- BBa_J04450 in pSB1A3
- BBa_J04450 in pSB1T3
- BBa_J04450 in pSB1C3
- BBa_J04450 in pSB1K3
- AmiC
- P-RBS
17.08.2012
- Restriction
- GroES (EcoRI/SpeI)
- Ligation
- GroES in pSB1C3
- Transformation
- GroES in pSB1C3
- Test restriction
- P-RBS (EcoRI/PstI)
- BBa_J04450 in pSB1A3 (EcoRI/PstI)
- BBa_J04450 in pSB1T3 (EcoRI/PstI)
- BBa_J04450 in pSB1C3 (EcoRI/PstI)
- BBa_J04450 in pSB1K3 (EcoRI/PstI)
- 1% Agarose gel
- 1% Agarose gel
Week 7
20.08. - 26.08.2012
20.08.2012
- PCR
- Backbone (primer MS24 + MS25)
- ⇒ template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
- 1% Agarose gel
- Transformation
- BBa_JH023
- Inoculation
- GroES
- P-RBS
- GFP-T
- Enhancer 3,4
- AmiC
- P-RBS alt
- GFP-T alt
21.08.2012
- Results
- colonies of BBa_JH023
- inoculations except AmiC and P-RBS alt successful
- PCR
- pSB1A3-BBa_J04450 (template DNA)
- pSB1T3-BBa_J04450 (template DNA)
- pSB1C3-BBa_J04450 (template DNA)
- pSB1K3-BBa_J04450 (template DNA)
- used primer --> MS24 + MS25
- initial denaturation 98°C - 5min
- denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
- 72°C - 5min --> 4°C - 8
- miniprep
- GroES, Enh 3/4 (+old), P-RBS (+old), GFP-T (+old)
- Test restriction
- of miniprep
- over night, 37°C
- 1% Agarose gel
- Inoculation
- E.coli BBa_I14032
- over night, 37°C
22.08.2012
- PCR
- CFP (MS16 + MS17)
- mRFP (MS14 + MS15)
- initial denaturation 98°C - 5min
- denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 35 times
- 72°C - 5min --> 4°C - 8
- 1% Agarose gel
- miniprep
- of LacIQ
- Test restriction
- of LacIQ with EcoRI and PstI
- 1% Agarose gel
- 1% Agarose gel
- 1% Agarose gel
- 1% Agarose gel
- test restriction of Enhancer (->21.08.12), gel 2%
- watch picture
- test restriction of Enhancer and P-RBS (->21.08.12), gel 2%
- watch picture
23.08.2012
- PCR
- pSB1K3-BBa_J04450 (template DNA)
- pSB1T3-BBa_J04450 (template DNA)
- used primer --> MS24 + MS25
- initial denaturation 98°C - 5min
- denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
- 72°C - 5min --> 4°C - 8
- 1% Agarose gel
- Restriction
- GixR (EcoRI/PstI)
- Ligation
- GixR in pSB1C3
- Transformation
- GixR in pSB1C3
24.08.2012
- PCR
- pSB1A3-BBa_J04450 (template DNA)
- pSB1T3-BBa_J04450 (template DNA)
- pSB1C3-BBa_J04450 (template DNA)
- pSB1K3-BBa_J04450 (template DNA)
- initial denaturation 98°C - 5min
- denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 30 times
- 72°C - 10min --> 4°C - 8
- primer: MS22 + MS23
- 1% Agarose gel
- Restriction
- RBS (XbaI/PstI)
- CFP (EcoRI/PstI)
- mRFP (EcoRI/PstI)
- Ligation
- mRFP and CFP in pSB1C3
- Transformation
- mRFP and CFP in pSB1C3
Week 8
27.08. - 02.09.2012
27.08.2012
- Results
- CFP: 1 colony
- CFP (neu): Colonies
- mRFP: colonies
- mRFP (neu): no colonies
- PCR
- template: backbones (pSB1K3/T3/C3/A3)
- parameters: 98°C 5min, >> 98°C 1min, 70°C 30s, 72°C 2.5min << x30, 72°C 5min, 4°C 8
- primer: MS22 + MS23
- colony-PCR
- parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C 8
- primer: MS18 + MS19
- Agarose gel of backbone PCR
- no result
- elution of the backbones of Friday the 24th
- 1% Agarose gel
- 1% Agarose gel
- Inoculation
- pSB1C3_...
- mRFP clone 20,24
- CFP clone 14,15,17,18
- gix clone 1,2
- 5ml LB, over night, 37°C
28.08.2012
- production of competent TOP10
- PCR
- Enhancer, Terminator, Gix, P-RBS
- parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
- primer: MS20 + MS21
- 1% Agarose gel
- Transformation
- [http://partsregistry.org/Part:BBa_I0500 pBad/araC] (plate:1, Well:N14)
- pSacB
- over night, 37°C
- miniprep
- GixR (1,2)
- CFP (14,15,17,18)
- mRFP (20,24)
- alle in pSB1C3
29.08.2012
- Results
- Transformation: no colonies
- PCR
- Terminator, Gix, Enhancer, P-RBS
- diluted templates: Terminator, P-RBS, Gix 1 --> 1:10
- Enhancer, Gix 2 --> 1:20
- parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
- primer: MS20 + MS21
- 1% Agarose gel
- 3AA
- CFP, mRFP with Terminator
- 2µl DNA
- 1µl SpeI
- 2µl Tango
- 15µl dH2O
- --> 1h at 37°C
- +1µl EcoRI
- +2,5µl Tange
- --> 1h at 37°C
- --> inactivation for 20min at 80°C
- Ligation
- 3AA products in pSB1T3
- 18.5µl H2O
- 2.5 µl Buffer
- 1µl Insert CFP or mRFP (EcoRI + SpeI)
- 1µl Insert Terminator (BBa_B0010) (XbaI + PstI)
- 1µl pSB1T3 (EcoRI + PstI)
- 1µl Ligase
- --> 1h at RT
- Transformation
- pBad --> TOP10 + pSB2K3
- CFP-T, mRFP-T --> TOP10 + pSB1T3
- Sequencing
- CFP_fwd
- CFP_rev
- mRFP_fwd
- mRFP_rev
- P-RBS_fwd
- GixR_fwd
- GixR_fwd
- Enhancer_fwd
30.08.2012
- Results
- Transformation: no colonies --> testing Trafo with BBa_J04450 A/K/T/C
- 2 colonies at pBad-Trafo from 28.08.2012 --> liquid culture for miniprep
- 1 colony at pSacB-Trafo from 28.08.2012 --> tested on succrose-sensitivity
31.08.2012
- PCR
- SacB
- Parameters No.1: 95°C 5', >>95°C 30", 60°C 30", 72°C 1'<< x30, 72°C 5', 4°C
- Parameters No.2: 95°C 5', >>95°C 30", 72°C 1.5'<< x30, 72°C 5', 4°C
- Primer MS26 + MS27
- 1% Agarose gel
- Ligation
- 3AA (CFP-T, mRFP-T)
- watch 29.08.
- used vector: pSB1K3/T3
- Transformation
- of ligation in TOP10
- miniprep
- pBad/araC K.1+2
- restriction
- GixR rehybridization
- cut with EcoRI and PstI
- ligation
- 1µl GixRhyb E+P
- 1µl pSB1C3 E+P
- 1µl Th-Pol
- 2µl Buffer
- 15µl dH2O
- Transformation
- ligation + TOP10 --> LB(cam)
- Results of test-trafo
- Amp, Km, Cm OK --> Km with fewer colonies
- Tet --> hardly colonies
- --> comp.TOP10 obviously all right, exclusion of Tet in future work
Week 9
03.09. - 09.09.2012
03.09.2012
- Results
- Transformation
- GixR in C3 --> no colonies
- CFP-T/mRFP-T in C3(31.8.) --> no colonies
- CFP-T/mRFP-T in T3(31.8.) --> many but small colonies
- CFP-T/mRFP-T in T3(29.8.) --> less colonies
- colony-PCR
- of colonies (CFP-T, mRFP-T)
- parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
- primer MS18 + MS19
- Agarose gel
- 6x colony pT3-CFP-T from 29./31.8.
- 6x colony pT3-mRFP-T from 29./31.8.
- pC3-CFP (14)
- pC3.mRFP (24)
- 1% Agarose gel
04.09.2012
- Results
- Transformation
- CFP-T/mRFP-T in T3
- 2 µl -> 5-6 colonies
- 30 µl -> 1-2 colonies
- Gix R in C3 (3.9) --> 4 colonies
- colony-PCR
- GixR
- CFP-T
- mRFP-T
- parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
- primer MS20 + MS21
- gel extraction
- P108 (E/S)
- RBS (X/P)
- ligation
- SacB1 + pSB1C3
- P-RBS + pSB1C3
- Transformation
- ligation + TOP10 --> LB(cmp)
- 1% Agarose gel
- Inoculation
- pBad/araC
- 5ml LB, over night, 37°C
- 1% Agarose gel
- 1% Agarose gel
05.09.2012
- Results
- Transformation
- SacB in C3
- P-RBS in C3
- colony PCR
- SacB in C3
- P-RBS in C3
- parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
- primer MS20 + MS21
- miniprep
- Gix in C3
- pBad
- Results
- Transformation
- CFP-T (03.09) --> 10 colonies
- colony PCR
- P108-RBS, SacB, GixR
- parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
- primer MS19 + MS20
- 1% Agarose gel
- 1% Agarose gel
- restriction
- pSB1A3 (E/P)
- pSB1K3 (E/P)
- pSB1C3 (E/P)
- pSB1T3 (E/P)
- GFP-T K3 (X/P)
- GFP K3 (X/P)
- CFP C3 (X/P)
- mRFP C3 (X/P)
- AmiC A (E/S)
- GroES A (E/S)
- Terminator (X/P)
- Inoculation
- pRBS
- SacB
- mRFP-Terminator
- 5ml LB, over night, 37°C
06.09.2012
- miniprep
- pRBS
- SacB
- mRFP-Terminator
- ligation
- AmiC (E/S) + GFP (X/P) + pSB1T3 (E/P)
- AmiC (E/S) + RFP (X/P) + pSB1T3 (E/P)
- AmiC (E/S) + GCFP (X/P) + pSB1T3 (E/P)
- AmiC (E/S) + Terminator (X+P + pSB1T3 (E/P)
- GroES (E/S) + GFP (X/P) + pSB1T3 (E/P)
- GroES (E/S) + RFP (X/P) + pSB1T3 (E/P)
- GroES (E/S) + CFP (X/P) + pSB1T3 (E/P)
- GroES (E/S) + Terminator (X/P) + pSB1T3 (E/P)
- PCR of miniprep
- SacB in pSB1C3
- p-RBS in pSB1C3
- terminator in pSB1T3
- parameters: 98°C 5min, >> 98°C 30 sec, 72°C 1,5min << x30, 72°C 5min, 4°C ∞
- primer MS20 + MS21
- 1% Agarose gel
- transformation
- RBS-Gin T3
- amiC A3
- groES A3
- groES-Terminator T3
- groES-GFP T3
- groES-CFP T3
- groES-mRFP T3
- amiC-Terminator T3
- amiC-GFP T3
- amiC-CFP T3
- amiC-mRFP T3
07.09.2012
- restriction (6.9.)
- ligation
- AmiC in pSB1K3
- GroES in pSB1K3
- mRFP in pSB1K3
- Terminator in pSB1K3
- CFP in pSB1K3
- GFP in pSB1K3
- GFP+Terminator in C3
- Enhancer in K3
- transformation
- Ligation
- AmiC
- GroES
- RBS-Gin
- miniprep
- CFP-Terminator
- Sequencing
- mRFP T3
- P-RBS K12
- P-RBS K15
- SacB K6
- SacB K6
Week 10
10.09. - 16.09.2012
10.09.2012
- Restriction
- Gin-RBS (01.08)
- Ligation
- Gin-RBS in pSB1C3
- Transformation
- Gin-RBS in pSB1C3
- Hybridisation
- GixR
- 98 °C 20 min
- minus 1 °C to 4 °C in 2 min
- Ligation and Transformation
- GixR (X+S) + pSB1K3 (X+S)
- Inoculation (Tetracycline)
- GroES-mRFP
- GroES-GFP
- GroES-CFP
- amiC-mRFP
- amiC-GFP
- amiC-CFP
- 5ml LB, over night, 37°C
- Inoculation (Kanamycine)
- GroES-GFP
- amiC-mRFP
- amiC-GFP
- GroES-Terminator
- Enhancer
- 5ml LB, over night, 37°C
- Inoculation (Chloramphenicol)
- GFP-Terminator
- 5ml LB, over night, 37°C
- Inoculation (Ampicillin)
- Terminator
- 5ml LB, over night, 37°C
11.09.2012
- microscopy-screening
- GroES-GFP ⇒; no fluorescence
- GroES-CFP ⇒; no fluorescence
- AmiC-mRFP ⇒; no fluorescence
- AmiC-GFP ⇒; no fluorescence
- colony-PCR
- GixR
- RBS-Gin
- Terminator
- parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
- primer MS20 + MS21
- 3A-Assembly (digestion, ligation, transformation)
- GroES (E/S) + Terminator (X/P) + pSB1K3 (P/E)
- AmiC (E/S) + Terminator (X/P) + pSB1K3 (P/E)
- plasmid-prep and control-digestion (E/P)
- GroES-GFP
- GroES-CFP
- AmiC-mRFP
- AmiC-GFP
- GroES-Terminator
- Enhancer
- GFP-Terminator
- Terminator
12.09.2012
- Results
- Transformation
- - P-RBS --> colonies
- - pB10 --> no colonies
- - RBS (B0030) --> colonies
- - Gro-ES/ AmiC-Terminator --> no colonies
- colony-PCR
- Gix (2. try)
- Gin-RBS (2. try)
- RBS (B0030)
- P-RBS (108)
- parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
- primer MS20 + MS21
- Agarose gel
- Gin-RBS (5x)
- Gin (1x)
- Gix (5x)
- RBS (6x)
- P-RBS (6x)
- 1% Agarose gel
- 1% Agarose gel
- Restriction (miniprep 11.09.12)
- gr-GFP
- gr-CFP
- gr-T K3
- enhancer K3
- am-RFP-K3
- am-GFP-K3
- GFP-T-C3
- terminator A2
- 1% Agarose gel
- 1% Agarose gel
- Transformation
- groES-T
- 2 Transformations:
- - One with a 1:10 dilution of Terminator from ligation,
- - another one with a 1:100 dilution of Terminator from ligation
- 2 Transformations:
- amiC-T
- 2 Transformations:
- - One with a 1:10 dilution of Terminator from ligation,
- - another one with a 1:100 dilution of Terminator from ligation
- 2 Transformations:
- PCR
- Gin-RBS
- 3 reactions:
- -One using High Fidelity (HF) Reaction Buffer,
- -one using GC-Buffer
- -and another one using self-made reaction buffer
- 3 reactions:
- Primer used : MS12 + MS13
- Template DNA : E. coli C600 chromosomal DNA
13.09.2012
- Results
- Transformation
- - pBAD (11.09.12) --> no colonies
- - AmiC-T (11.09.12) --> 2 x 2 colonies
- - GroES-T (11.12.09)--> 2 colonies
- colony-PCR
- AmiC-T & GroES-T (transformation 11.09.12)
- parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
- primer MS20 + MS21
- 1% Agarose gel
- Gel extraction
- RBS-Gin (HF) --> 51 ng/µl
- RBS-Gin (GC) --> 69 ng/µl
- RBS-Gin (SM) --> 56 ng/µl
- Ligation
- GroES-T & AmiC-T in pSB1K3
- Restriction (preparative digest)
- GroES (A module)
- with SpeI
- AmiC (A module)
- with SpeI
- mRFP-C3 (B module)
- with EcoRI and XbaI
- CFP-C3 (B module)
- with EcoRI and XbaI
- Gin-RBS
- HF-PCR-Product, GC-PCR-product and SM-PCR-product
- with EcoRI and PstI
- HF-PCR-Product, GC-PCR-product and SM-PCR-product
- 1% Agarose gel
14.09.2012
- Results
- Transformation
- AmiC-T: no colonies
- GroES-T: no colonies
- Restriction
- 2nd Digestion of A-modules (see digest from yesterday) additionally with EcoRI
- Ligation
- Gin-RBS with pSB1C3
- 3 reactions:
- one with RBS-Gin HF-PCR-Product
- one with RBS-Gin GC-PCR-Product
- one with RBS-Gin SM-PCR-Product
- 3 reactions:
- Inoculation
- Gix-K3 (clone 3 and 4)
- P-RBS-C3 (clone 1 and 3)
- RBS (BBa_B0030) [clone 1 and 3]
- 1% Agarose gel
- Gel extraction
- GFP
- mRFP
- 1% Agarose gel
- Gel extraction
- Terminator clone 48
- Transformation
- RBS-Gin(HF)-pSB1C3
- RBS-Gin(GC)-pSB1C3
- RBS-Gin(SM)-pSB1C3
Week 11
17.09. - 23.09.2012
17.09.2012
- Restriction
- AmiC
- GroES
- CFP
- SacB
- 1% Agarose gel
- Restriction
- CFP-C3
- SacB (2nd try)
- Resuspend
- BBa_B001J
- Restreak
- RBS-Gin (HF)
- RBS-Gin (GC)
- RBS-Gin (SM)
- DNA isolation
- CFP-C3 (Miniprep28.08.12)--> 47,3 ng/µl
- Miniprep
- GixR pSB1K3 --> 50,5 ng/µl, 144,7 ng/µl
- RBS(new)B0030 pSB1A2 --> 726,2 ng/µl, 242,6 ng/µl
- P108-RBS pSB1C3 --> 200,5 ng/µl, 177,8 ng/µl
- 1% Agarose gel
- Transformation
- pSB2K3-pBAD & pSB1-A2-TT
- 1% Agarose gel
- CFP-C3 (restriction 2nd try)
- SacB-Cs (restriction 2nd try)
18.09.2012
- Results
- Transformation
- 2x Terminator --> colonies
- pBAD --> no colonies
- Colony PCR
- RBS-Gin
- 2x Terminator
- parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
- primer MS20 + MS21
- 1% Agarose gel
- Sequencing
- SacB
- Gel extraction
- CFP-C3 E+X
- Ligation
- am-G: amiC E+S & GFP-A3 E+X
- am-C: amiC E+S & CFP-C3 E+X
- am-R: amiC E+S & mRFP-C3 E+X
- gro-G: groES E+S & GFP-A3 E+X
- gro-C: groES E+S & CFP-C3 E+X
- gro-R: groES E+S & mRFP-C3 E+X
- Transformation
- am-G
- am-C
- am-R
- gr-G
- gr-C
- gr-R
- pBAD
19.09.2012
- Results
- Transformation
- am-G --> colonies
- am-C --> colonies
- am-R --> colonies
- gr-G --> colonies
- gr-C --> colonies
- gr-R --> colonies
- pBAD --> no colonies
- Colony PCR
- RBS-Gin (from 17.09.12)
- 2x Terminator B0017 (from 17.09.12)
- parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
- primer MS20 + MS21
- --> Amplification failed
- Transformation
- pBAD
- Microscopy-screening
- AmiC-GFP --> fluorescence
- GroES-GFP --> fluorescence
- AmiC-CFP --> fluorescence
- GroES-CFP --> fluorescence
- AmiC-mRFP --> fluorescence
- GroES-mRFP --> fluorescence
- Inoculation
- RBS-Gin
- TT (B0017)
- AmiC-GFP
- GroES-GFP
- AmiC-CFP
- GroES-CFP
- AmiC-mRFP
- GroES-mRFP
20.09.2012
- Miniprep
- RBS-Gin
- TT (B0017)
- AmiC-GFP
- GroES-GFP
- AmiC-CFP
- GroES-CFP
- AmiC-mRFP
- GroES-mRFP
- Restriction (E/P)
- TT(B0017)
- RBS-Gin
- 1% Agarose gel
- Restriction
- SacB ((XbaI + PstI) & (EcoRI + XbaI)
- P-RBS (EcoRI + SpeI) & (SpeI + PstI)
- 1% Agarose gel
- Inoculation
- GroES-GFP
- GroES-CFP
- GroES-mRFP
21.09.2012
- Restriction
- GFP-fusion (EcoRI + SpeI)
- TT BBa_0017 (EcoRI + XbaI)
- 1% Agarose gel
- Gel extraction
- GFP-Fusion
- pSB1A2 (backbone GFP-Fusion)
- TT
- SacB
- Ligation
- GFP bb Enhancer
- GFP TT
- GroES TT
- Transformation
- GFP bb Enhancer
- GFP TT
- GroES TT
- Promotor-RBS (17.09.12)
- Sequencing
- RBS-Gin
- Microscopy-screening
- AmiC-GFP --> Channel: DIC, Green, Red, Blue
- GroES-GFP --> Channel: DIC, Green, Red, Blue
- AmiC-CFP --> Channel: DIC, Green, Red, Blue
- GroES-CFP --> Channel: DIC, Green, Red, Blue
- AmiC-mRFP --> Channel: DIC, Red
- GroES-mRFP --> Channel: DIC, Green, Red, Blue
22.09.2012
- Inoculation
- GroES-TT pSB1A2
- GFP-TT pSB1A2
- Enhancer
- P108-RBS pSB1C3