Team:BYU Provo/Team Thermosensor/The Beginning

Week Feb. 21 – 27

• Feb. 22
  • Made PCR for thermosensor will check prouct tomorrow
• Feb. 23
  • PCR products checked, thermosensor appears to have been amplified correctly
  • pPLAT extracted via qiaprep plasmid prep protocol

Week Feb. 28 – Mar. 6

• Feb. 28
  • Phusion PCR for GFP in Thermosensor system
    • ~35 uL ddH2O
    • 10uL 5X phusion buffer
    • 1.5uL 10mM dNTP’s
    • 1uL each primer
    • 1uL of diluted template DNA
    • 0.5 uL phusion polymerase
  • Purified PCR samples according to Qiagen Kit
  • Restriction Digest of insert and vector inserting thermosensor into pPLAT
    • PCR product(insert) digestion (50uL rxn)
      • ~14uL H2O
      • 5uL 10X NEB buffer
      • 0.5 uL 100X BSA
      • 30uL DNA sample
      • 1-2uL each restriction enzyme
    • Vector Digestion (50uL rxn)
      • ~14uL H2O
      • 5uL 10X NEB buffer
      • .5 uL 100X BSA
      • 30uL DNA sample
      • 1-2 uL each restriction enzyme
        • Placed reactions in 37o bath for 1.5 hr+

• Mar. 2
  • Restriction Digests actually done today
  • Running a gel of purified thermoswitch and purified GFP
    • (thermoswitch in 3rd well and GFP in 5th well)

• Mar. 4
  • Product from redo thermoswitch PCR

Week Mar. 21 – 27

• Mar. 23
  • Low Melt gel ran on vector and insert, bands cut out and gel cut-outs melted at 65oC
  • Received pGLO from Dr. Grose on Monday to be transformed into competent cells to act as a positive control for GFP
  • pPLAT can be transformed into competent cells(DH5-a) as a neg. control
    • (GFP was more widely used by the OxyR team so they did the transformation of pGLO into competent cells)
  • Began ligation for pPLAT and pBAD cut at BamHI and EcoRI
    • 6.5 uL H2O
    • 1.5uL 10X ligase buffer
    • 1 uL T4 DNA ligase
    • 3uL vector
    • 3uL insert (didn’t put insert in control)