IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/07/30: Difference between revisions

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==Liquid Culture (Osnat's strain)==
Each 10ml + 10 uL antibiotics (if any)
*ColE7
*ColE2
*pDew-E2 (Amp)
*pDew-E7 (Amp)
From the paper, the bacteria strain is B2B1011 which has Str-resistance
==Digestion of pSG.005 and pSG.014==
==Digestion of pSG.005 and pSG.014==


Revision as of 09:50, 30 July 2012

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Liquid Culture (Osnat's strain)

Each 10ml + 10 uL antibiotics (if any)

  • ColE7
  • ColE2
  • pDew-E2 (Amp)
  • pDew-E7 (Amp)

From the paper, the bacteria strain is B2B1011 which has Str-resistance

Digestion of pSG.005 and pSG.014

PSG.005 (vector) PSG.014 (insert)
Green Buffer 4 uL 4 uL
SpeI 1 uL -
PstI 1 uL 1 uL
XbaI - 1 uL
Fast AP 1 uL -
Plasmid 5 uL (+/- 1000ng) 20 uL (+/- 500 ng)
H2O 28 uL 14 uL

Ligating oligos

  1. iSG.009 + iSG.010 = pSG.004 (pTet)
  2. iSG.011 + iSG.012 = pSG.015 (constitutive promoter; strength = 0.86)
  3. iSG.013 + iSG.014 = pSG.016 (constitutive promoter; strength = 0.51)

each of them in form digested with XbaI and PstI.

Protocol

Mix:

  • 2 μL 10 μM sense oligo
  • 2 μL 10 μM anti-sense oligo
  • 2 μL 10xPNK (polynucleotide kinase buffer A)
  • 2 μL 10mM ATP
  • 1 μL T4 polynucleotide kinase (PNK)
  • 10 μL distilled water

to give 20 μL total volume

Incubate at 37°C for 30 mins

Place in boiling water bath for 2 min., then remove water bath from the heat source and allow the reaction (still in the water bath) to cool to room temperature.

Ligation of pSG.005 and pSG.014

PSG.005 (negative control) PSG.008 (pSG.005+pSG.014)
Vector (+/- 25 ng) 2 uL 2 uL
Insert (+/- 25 ng) - 2 uL
Buffer 1 uL 1 uL
Ligase T4 0.5 uL 0.5 uL
Water 6.5 uL 4.5 uL


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