Jessica Karen Wong/Notebook/2007-7-25

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

• 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