Jessica Karen Wong/Notebook/2007-7-9

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Contents

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
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
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