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

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

• PCR
  • Template: 40ul PCR Supermix, .4ul of each primer, and .4ul of of N. gonorrhea DNA
    • NEW B: Part 1 of IgA beta-core IgAb-F and NEW_Mut_Pst1a-R
    • NEW C: Part 2 of IgA beta-core NEW_Mut_Pst1a-F and NEW_Mut2_Pst1b-R
    • NEW D: Part 3 of IgA beta-core NEW_Mut2_Pst1b-F and IgAb-R
  • Same protocol as 1.21.2008

• Solid Culture
  • Grew up samples from transformation on Amp/Cl plates overnight at 37C