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

 * 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