Jessica Karen Wong/Notebook/2007-7-25

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Contents

I2056

  • Heat shocked overnight digest w/ Mfe1 and Nsi1
  • PCR cleaned
  • Ligated into 1AK3
  • Transformed 3ul of ligation and plated
  • Was going to ligate into 3K3 also but ran out of 3K3 cut E/P

T9002

  • Heat Shocked overnight E0240 digest w/ Spe1 and Nsi1
  • PCR cleaned E0240
  • Ligated F2620-3K3 (2 samples) and F2620-1AK3
  • Transformed 3ul of each ligation and plated

Plasmids

  • Minipreped overnight of 1AK3
  • Digesting 1 sample with Eco and Pst and the other with Eco and Xba
  • Digesting 3K3 w/ Eco and Pst overnight
  • Also made an overnight of 3K3 from registry plate b/c ran out of uncut DNA too

PCR

Gel from 7-25
Gel from 7-25
  • Diluted P1010_Xba_R, P1010_Eco_F, P1010_Spe_F, I2055_Promoter, E0240_R to 40uM
  • Set up 1 100ul P1010 w/ Eco FWD and Xba REV
  • Set up 1 100ul P1010 w/ Spe FWD and Xba REV
  • Set up 1 100ul I2055 (new I2055_Promoter FWD and new E0240_R)
  • All rxn involve supermix and are at 53
  • Ran Gel loaded: lad, sp, I2055, ccdb-e/x, ccdb-s/x
    • Slightly blurry but all look the right size - CCDB's around 700 and I2055 around 1kb
  • Set up 24 overnight colony PCR's of I2055-3K3

Digests

  • Set up an overnight double digest of I2055 with Mfe1 and Nsi1 in buffer 2
  • Overnight double digest of P1010 w/ Eco and Xba tails cut E/X
  • Overnight double digest P1010 w/ Spe and Xba tails cut S/X
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