Jessica Karen Wong/Notebook/2007-8-2

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