Jessica Karen Wong/Notebook/2007-8-2

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Sequencing Redos

  • I2057-EX-VR failed again
  • I2057-ES-VF failed again
  • E0240-ES-VF forward is perfect until 260, all is fine except for the 1bp mutation around 700
  • I2056-2-F still has the 1bp insertion at around 20, rest is fine
  • I2056-3K3-1F has a 1bp deletion at about 60, rest is fine
  • I2055-3K3-1-F perfect until 758, full coverage and scarred

Made overnights of 4 new colonies of I2056 (#4-7), I2057-ES, I2057-EX

I2055

  • PCR cleaned overnight digest and RBS digests (B0030, 31, 32)
  • Has complete go ahead
  • Ligated:
    • I2055-NOT-EX closed
    • I2055-Not-SX closed
    • I2055-EX-B0032
    • I2055-SX-B0032
    • I2055-SX-B0030
    • I2055-SX-B0031

I2056 - overnight plates had no growth, seq was bad, will be sending in colonies 4-7 to seq

I2057

  • Minipreped I2057-ES and I2057-EX and digested E/S and E/X respectively
  • Didn't get enough DNA so making another overnight to use
  • Also overnighting to seq
  • Digested I2057-ES that had been sealed with Not1, cut E/S
  • Ligated PCR product cut either ES and EX w/ promoters
    • I2057-ES-R0040
    • I2057-EX-R0040
    • I2057-EX-J100
    • I2057-EX-J116
  • PCR cleaned I2057-ES E/S digest from today

Made new Kan plates

F2620

  • Ran gel of overnight PCR - looked good
  • PCR cleaned
  • F2620-3K3 plate from overnight had no growth
  • Digested PCR product Mfe/Xba in buffer 4
  • PCR cleaned digest
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