Jessica Karen Wong/Notebook/2007-7-9

Sequencing

 * Minipreped overnights with 8.5 water
 * Checking to make sure they're good: I2055-1AK3 (make sure constructed), E0240-1AK3 (see if scarred)
 * Checking to see what went wrong: T9002-3K3, E0240-3K3

3K3

 * Minipreped overnight culture from registry plate with EB
 * Digested E/P using 16ul DNA
 * PCR cleaned

I2057

 * BB forward primer came in
 * Diluted to 40umol w/ 485ul water
 * Did a 100ul prep PCR to insert BB site
 * Forward primer: BB_E0240_F
 * Rev primer: BB_Backbone
 * used Vent, 2:40 elongation time, and 53.5 degrees
 * Looked good on the gel
 * PCR cleaned
 * Set up an overnight double digest with Mfe1 and Nsi1
 * Used 4ul DNA and buffer 2

T9002-1AK3

 * Overnight transformation had 3 colonies
 * Made a 50ul cell suspension of each
 * Did a 10ul Colony PCR on each, 53 and 2min extension time

I2056

 * Set up an overnight scarring PCR
 * 100ul at 54C

Gel

 * Ran an analytic gel on the Biobricking PCR of E0240, the Colony PCR's of T9002, and the scarring PCR of I2057
 * Loaded: Space Ladder E0240 T9002(1-3) I2057
 * The I2057 is very bright and seems to be the right size
 * The others didn't work, retrying them