Jessica Karen Wong/Notebook/2007-7-17

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Sequencing

Made glycerols, minipreped, and sent for sequencing w/ VF and VR

  • E0240-1AK3 Colonies 1 and 2
  • I2055-1AK3 #4 and #16
  • E0240-3K3 #9
  • I2057-3K3 #2 and #4
  • I2055-3K3 #3
  • and 2 of Jason's 507 and 528

Also sent T9002-3K3 #12 for seq with VF, VR, and internal primers

PCR

  • Made 8 cell suspensions of I2055-3K3 and ran 10ul rxns of them w/ taq and a 1:30 ext time
  • PCR'ed I2056 to scar and add in promoter
    • Diluted I2056_R0040_F to 40 ul
    • Also used I2055_R
    • 1 100ul rxn at 53 w/ vent
  • PCRed E0240 portion of T9002 1 100ul rxn w/ vent at 54
  • Set up overnight BB PCR of E0240-3K3
    • 1 100ul w/ vent at 53.5, 2:30 ext time

F2620

  • PCR cleaned digests of F2620 (M/N), 1AK3 (E/P), 3K3 (E/P)
  • Ligated F2620-1AK3 and F2620-3K3
  • Transformed 3ul of each ligation and plated

Gels

Gradient T9002-E0240 PCR
  • Ran gel of overnight gradient pcr of T9002_E0240 (gel 1) - looked good
  • Ran gel of today's pcr's
    • Loaded: Lad, Sp, I2056, Sp, I2055-3K3 (colonies 1-8)
  • Made an overnight of Colony #1 to sequence
Scar and Colony PCRs