Jessica Karen Wong/Notebook/2007-7-10

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  • Heat shocked overnight digest of I2057 with Mfe1/Nsi1
  • PCR cleaned I2057 digest
  • Ligated with both 1AK3 and 3K3
  • Transformed 3ul of each and plated on Kan


  • PCR cleaned overnight scarring PCR of I2056
  • Ran gel of I2056 and overnight colony PCR's
    • Nothing showed up the right size
  • Got sequencing in for I2056
    • Sequence only matched plasmid, none of the part was present
  • Discarded cleaned PCR
  • Rebuilding part: ligated R0040-J04650-1AT3, R0040-J04650-1AC3
  • Transformed 3ul of each and plated on appropriate antibiotic
  • Made new Chlor plates, running low


  • Ligated T9002-3K3 and transformed
  • Also spun down rest of T9002-1AK3 transformation and plated


  • Redid BioBricking PCR of E0240-1AK3 on a gradient
    • 12 10ul PCR's from 51c to 57c
    • Used Taq and too short elongation time 1:45
  • This product was not the right size (rightmost lanes)
    • Has some at 3kb and some at around 10kb, but should be 4kb
  • Redid BB PCR overnight on same gradient with Vent and proper 2:40 elongation time
  • Made new overnight of E0240-1AK3 b/c were running out of DNA
  • Religated and transformed E0240-3K3


  • Scarred product in 1AK3 had 5 colonies on the transformation plate
  • Colony PCR-ed all 5
  • Ran a gel and nothing showed up again (the first five lanes after the ladder)
  • Did a positive control with P1010 from the registry to make sure we're doing colony PCR right
    • 1 10ul with VF and VR and extension time 1:30

Gel from 7/10/07