Jessica Karen Wong/Notebook/2007-6-21

To Do

• Get Jason to log in to point mutate I2054
• Make plan for next week
• Digest backbones, J04650, B0032, R0040
• PCR T9002, E0240

Digest

• See protocol
• Digested plasmids 1AT3, 1AK3, and 1AC3 with EcoR1 and Pst
  • Used 3ul of AT3 and AC3 plasmid and 4ul AK3 to insure 1 ug of DNA
  • Used EcoR1 buffer
• Digested B0032 and R0040 with EcoR1 and Spe
  • Used 2ul of B0032 DNA 3ul of R0040 DNA
  • Used EcoR1 buffer
• Digested J04650 with Xba and Pst
  • Used 5ul of prepped DNA
  • Used buffer 3 as suggested by NEB for the XbaI/PstI digests
• Incubated at 37C overnight

PCR

• Did a 100ul preparatory PCR on T9002 and E0240
• Based on Endy:Colony PCR
  • 95.25 ul PCR Mix
    • 25ul Thermo Polymerase Buffer
    • 25ul dNTP's (10x)
    • 3.75ul Rev Primer (40uM)
    • 2.5ul Taq Polymerase
    • 183 Water
  • 1 ul DNA (3x dilution)
  • 3.75 ul forward primer (because T9002 and E0240 have different forward primers but the same reverse primer)
• Dilute primers to 40 uM
  • Added 730ul water to E0240F
  • Added 821ul to T9002F
  • Added 925ul to E0240R
• Did a 3x dilution of DNA samples

Sequencing

• Nishant prepped and sent I0255 to be sequenced