Team:Frankfurt/Notebook

From 2012.igem.org

(Difference between revisions)
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=Labwork=
=Labwork=
==May and June 2012==
==May and June 2012==
-
* Arrangements for labwork
+
* Arrangements for labwork <br>preparation of competent cells (E.coli, S.cerevisiae), agarose plates (LB, YEPD, SCD-ura,…), medium for E.coli and S.cerevisiae
-
<space>preparation of competent cells (E.coli, S.cerevisiae), agarose plates (LB, YEPD, SCD-ura,…),medium for E.coli and S.cerevisiae
+
* Purchasing of the equipment (reaction tubes, glass bottles, pipette tips,..)
* Purchasing of the equipment (reaction tubes, glass bottles, pipette tips,..)
* Primer design
* Primer design
 +
==July 2012==
 +
* Plasmid isolation of p426, p423, pUD8e, pUD22e from E.coli
 +
 +
* Isolation of chromosomal DNA of CEN.PK2-1C
 +
 +
* Trials to get the genes, promoters and terminators via PCR
 +
==August 2012==
 +
* PCR of the genes, promoters and terminators<br>all genes (without KO and KAH), promoters and terminators could be amplified
 +
{|class wikitable float:right
 +
|-
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!colspan=4|Templates||Amplified DNA Fragments
 +
|-
 +
|synthesized sequence of HMG-CoA||HMG-CoA
 +
|-
 +
|synthesized sequence of GGPPS||GGPPS
 +
|-
 +
|synthesized sequence of Cps/Ks||CPS/KS
 +
|-
 +
|chromosomal DNA of CEN.PK2-1C||ERG20
 +
|}
 +
* Linearization of p426 and p423 with SpeI and XhoI
 +
* Biobrick production of the genes HMG-CoA, ERG20, Cps/Ks
 +
 +
- restriction of 3 µg of the genes with EcoRI and PstI
 +
 +
- ligation of biobrick genes with linear pSB1C3
 +
 +
- transformation of the ligation in E.coli
 +
 +
- plasmid isolation of E.coli clones
 +
 +
- control restriction of biobrick plasmids with EcoRI and SpeI
 +
==September 2012==
 +
==October 2012==
=Methods and Protocols=
=Methods and Protocols=
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pH has to be regulated with KOH to pH=6.3 <br>
pH has to be regulated with KOH to pH=6.3 <br>
-
* contains no His, Leu, Trp and Uracil <br> ** addition of this components depents on the respective selection medium
+
* contains no His, Leu, Trp and Uracil <br> ** addition of this components depents on the respective selection medium
{|class=wikitable float:right
{|class=wikitable float:right
|-
|-

Revision as of 11:19, 20 September 2012

Frankfurt logo.png
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You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Contents

Labwork

May and June 2012

  • Arrangements for labwork
    preparation of competent cells (E.coli, S.cerevisiae), agarose plates (LB, YEPD, SCD-ura,…), medium for E.coli and S.cerevisiae
  • Purchasing of the equipment (reaction tubes, glass bottles, pipette tips,..)
  • Primer design

July 2012

  • Plasmid isolation of p426, p423, pUD8e, pUD22e from E.coli
  • Isolation of chromosomal DNA of CEN.PK2-1C
  • Trials to get the genes, promoters and terminators via PCR

August 2012

  • PCR of the genes, promoters and terminators
    all genes (without KO and KAH), promoters and terminators could be amplified
TemplatesAmplified DNA Fragments
synthesized sequence of HMG-CoAHMG-CoA
synthesized sequence of GGPPSGGPPS
synthesized sequence of Cps/KsCPS/KS
chromosomal DNA of CEN.PK2-1CERG20
  • Linearization of p426 and p423 with SpeI and XhoI
  • Biobrick production of the genes HMG-CoA, ERG20, Cps/Ks

- restriction of 3 µg of the genes with EcoRI and PstI

- ligation of biobrick genes with linear pSB1C3

- transformation of the ligation in E.coli

- plasmid isolation of E.coli clones

- control restriction of biobrick plasmids with EcoRI and SpeI

September 2012

October 2012

Methods and Protocols

Plasmid Preparation

Plasmid Preparation of E. coli (Mini Preparation)

Plasmid Preparation of Saccharomyces cerevisia

Transformation

Yeast Transformation

E. coli Transformation

PCR

Culture Medium

Full Medium (YEPD) for Yeast
Yeast Extract1 % (weight/volume)
Pepton2 % (w/v)
Glucose2 % (w/v)
Synthetic Complete Medium (SC) for Yeast
Yeast Nitrogen Base0.17 % (w/v)
Ammoniumsulfate0.5 % (w/v)
Glucose2 % (w/v)
Amino Acid Mix*50 ml/l
Histidin**0.25 mM
Tryptophan**0.19 mM
Leucin**0.35 mM
Uracil**0.44 mM

pH has to be regulated with KOH to pH=6.3

  • contains no His, Leu, Trp and Uracil
    ** addition of this components depents on the respective selection medium
SOC-Medium for Regeneration of transformed Escherichia coli`s after Electroporation
Trypton2 % (w/v)
Yeast Extract0.5 % (w/v)
NaCl10 mM
KCl2,5 mM
MgCl210 mM
MgSO410 mM
Glucose20 mM

pH has to be regulated to pH=6.8-7.0

Full Medium (LB) for E. coli
Yeast Extract0.5 % (w/v)
Trypton1 % (w/v)
NaCl0.5 % (w/v)

pH has to be regulated with NaOH to pH=7.5

Every cluture medium has to be autoclaved to be sterile.

Agar Plate

LBampicillin-Agar

Add 2 % agar to LB-medium. After autoclaving and cooling-down to 60 °C steril ampicillin is added. Plates were poured.

SCD-Agar

Add 2 % agar to SCD-medium. After autoclaving and cooling-down steril amino acid solution is added. Dependent on the respective selective medium Histidin (0.25 mM), Trypthophan (0.19 mM), Uracil (0.44 mM) or Leucin (0.35 mM) are added. Plates were poured.

YEPDG418-Agar

Add 2 % agar to YEPD-medium. After autoclaving and cooling-down sterile G418 (final concentration 2g/l) is added. Plates were poured.

Gel Electrophoresis

Agarose Gel (1x)
TAE puffer1x
Agarose1 % (w/v)

Solve agarose in TAE by boiling it. After cooling-down to 55-60 °C gel is poured.

TAE Puffer (50x) for Gel Electrophoresis
EDTA18,6 g
Tris242g
Glacial Acetic Acid57,2 ml
Purified Water1000ml

pH has to be regulated with glacial acetic acid to pH=8.

Kit

PCR Purification Kit
Gel Extraction Kit
Midi Plasmid Preparation Kit

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