IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/08/07: Difference between revisions

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*MAGE
*MAGE
*Top 10
*Top 10
was diluted 100x, plated 20 uL with beads
were diluted 100x, plated 20 uL with beads


then 5 uL drops of
then 5 uL drops of

Revision as of 02:02, 13 August 2012

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Ligation

Products

Product Description Vector (digested with) Insert (digested with) Antibiotic resistance
PSG.003 (-) PSB1A2 pLac, negative control PSG.003 (S,P) - Amp
PSG.003+pSG.014 pSB1A2 pLac ColE2Imm PSG.003 (S,P) PSG.014 (X,P) Amp
PRG.037 (-) PLac (Denis), negative control PRG.037 (S,P) - Amp
PRG.037 + pSG.014 pSB1A2 pLac (Denis) ColE2Imm PRG.037 (S,P) PSG.014 (X,P) Amp
RBS+V (-) (-) control of the (+) control PSB1A2+RBS (S,P) - Amp
RBS+V+RFP (+) control PSB1A2+RBS (S,P) RFP (X,P) Amp

Protocol

Vector 0.5 uL
Insert 8 uL (or 0 uL for the negative controls)
H2O 0 uL (or 8 uL for the negative controls)
Fermentas T4 Ligase Buffer 10x 1.0 uL
Fermentas T4 Ligase enzyme 0.5 uL
Total 10 uL

Incubate at room temperature for 10min.

Transformation

  • Transformed 2 uL and 8 uL of each ligation product
  • No colony on pSG.003 (-), pSG.003+pSG.014
  • Some colonies on pRG.037+pSG.014 → liquid culture 5 colonies

Colicin experiment

Saturated liquid culture of different type of cells:

  • Wild type MG1655
  • Turbo
  • MAGE
  • Top 10

were diluted 100x, plated 20 uL with beads

then 5 uL drops of

  • ColE2
  • ColE7
  • the same type of cell

added to each plate

Results

ColE2 seems ok. ColE7??


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