Jessica Karen Wong/Notebook/2007-6-21

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Contents

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
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