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

Gel from 7/9/07

• 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