User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/23

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Cellular Adhesion

  • Gel
    • Ran 1% gel for 25 minutes at 95V with samples (5ul sample with 2ul of loading dye)
    • Not a great gel, but the smudges around 100 bp look promising
    • Extra band in GCN4 sample
log2-ladder, 6His, FLAG, myc, HA, GCN4
  • PCR Purification
    • Used MinElute columns to purify the five tags and one zipper from 1.22 PCR
    • Eluted in 10ul of water
  • Digest
    • Ran on PCR purification above
    • Template for tags: 3ul DNA (each), 2ul NEB2, .5ul EcoR1, .5ul Pst1, rest water (20ul rxn)
    • Thermocycler protocol: 1hr@37C, 20mins@80C
  • Ligation
    • Ran on digest samples above
    • Template for assembly: .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
    • Protocol: 30mins@roomtemp,10mins@65C
  • Transformation
    • 50ul of TOP10 cells on ice with 2ul of DNA for 30mins
    • 45secs heat shock at 42C using water bath
    • Added 300ul of SOC
    • Incubated for 1hr at 37C
    • Plated on Amp/Cl and grew up at 37C overnight
  • Solid Culture
    • Grew up samples from transformation on Amp/Cl plates overnight at 37C