IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-13

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Gel purification, Ligation and transformation of GeneArt Plasmids, natural KaiA into J04500 backbone

I gel purified the inserts pre-ligation, but the nanodrop gave extremely low (eg. negative) numbers. Then, I used an absolute ratio of 1:3 vector-insert instead of the normal molar ratio. DO NOT USE THIS PROTOCOL FOR SUBSEQUENT LIGATIONS!

Ligation for samples the nanodrop hates

  • BB vector J04500 \SP: 2.5uL
  • BB insert KaiA, B, or C \XP: 7.5uL

Protocol:

  • Mix above ratios + 10uL #1 + 1uL #3
    • BE sure to mix #1 (ligation buffer) well especially!
  • Sit at bench for ~30min
  • Put back on ice

Transformation

For the transformation I did 5 samples:

  • 1 exp. (50uL) natural KaiA
  • 1 exp. (50uL) synthetic KaiA
  • 1 exp. (50uL) synthetic KaiB
  • 1 exp. (50uL) synthetic KaiC
  • 1 (25uL) positive
  • 1 (25uL) negative

Ice incubation 30m, 30s@42C, 2m@ice, SOC@1h@37C. Plated on LBCarb. Should be ready around 6PM to see.

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