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From 2010.igem.org

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Revision as of 22:31, 25 July 2010

Johan

Colony PCR

• A colony PCR on the TAT CCP (N-version) was performed as the result from the last gel electrophoresis (insert date) was just a smear on almost all bands. The reason was most probably too much genomic DNA (too much colony picked), so less of the colonies was picked and the final volume was increased from 25 µl to 50 µl.

• A colony PCR on the LMWP CCP (N- & C-version) was performed as the result from Mimmis gel electrophoresis was just empty bands (insert date).

• TAT: colonies #1-41 +insert, #42-43 -insert.
• LMWP: colonies #1 -insert, #3-24 +insert.

• PCR reaction mix
  • 1 µl Pfu polymerase
  • 1 µl 10 mM dNTPs
  • 3 µl 10 µm f.primer (VF2)
  • 3 µl 10 µm r.primer (VR)
  • 5 µl Pfu buffer 10x
  • 32 µl H2O (miscalculation, should have been 37 µl)

• PCR program
  • 98 °C - 2 min
  • 35 cycles of
    • 98 °C - 10 sec
    • 55 °C - 15 sec
    • 72 °C - 30 sec
  • 72 °C - 5 min
  • 4°C - ∞