Jessica Karen Wong/Notebook/2007-7-31

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  • E0240-1AK3 - double Mfe piece went in backwards, discarding
  • E0240-3K3-E-S - Fwd failed, Rev shows a 1bp mutation around 700
  • E0240-3K3-E-X - Full coverage, mutation at same spot as in ES
  • I2055-3K3-1 - looks fine 1-95 and 208-end, has promoter and is scarred
  • I2055-3K3-2 - wrong, discarding
  • I2056-3K3-1 - Fwd failed, Rev shows scarring, waiting on redo to see if front is ok
  • I2056-3K3-2 - Mutated, discarding
  • I2057-3K3-ES - Fwd failed, 267-end looks ok
  • I2057-3K3-EX - Rev failed, 1-769 looks ok

Doing a site directed mutagenesis on BBed E0240's, giving up on getting devices into 1AK3


  • Minipreped overnight cultures of J23116 and J23100 - will save for later
  • Prepared Chemically Competent DB3.1 cells
  • BB PCRed I2055-3K3-1
    • Diluted BB_I2055-Fwd-X to 40uM
    • Made 1 100ul rxn w/ BB_R_Rev_S primer, and another with BB_R_Rev_E
  • Transformed and Plated:
    • I2057 E/S sealed w/ Not1 in DB3.1
    • I2057 E/X sealed w/ Not1 in DB3.1
    • 3 samples of F2620-3K3 in Top10
  • Digested: I2056-E-X w/ EcoR1 Xba1, I2056-E-X w/ Not1, I2056-S-X with Spe1 and Xba1, and I2056-S-X w/ Not1
    • Not digests in buffer 3, E/X and S/X digests in buffer 2