User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/03

Cellular Adhesion

• Miniprep
  • QIAGEN miniprep on liquid cultures from yesterday (leucine zippers)
  • Spun down for 10min at 3000rcf then followed protocol

• Spec
  • DNA containing Fos: 51.8 ng/ul
  • DNA containing JunB: 51.4 ng/ul
  • DNA containing GCN4: 90.0 ng/ul

Ran too long

Ran too long

• PCR
  • Conservative IgAbc F (B + C with D): 40ul PCR Supermix, .4ul IgAb-F and IgAb-R, 1ul ligation product
  • Conservative IgAbc R (C + D with B): 40ul PCR Supermix, .4ul IgAb-F and IgAb-R, 1ul ligation product
  • DNA containing Fos: 40ul PCR Supermix, .4ul Fos-F and Fos-R, 2ul DNA
  • DNA containing JunB: 40ul PCR Supermix, .4ul JunB-F and JunB-R, 2ul DNA
  • DNA containing GCN4: 40ul PCR Supermix, .4ul GCN4-F and GCN4-R, 1ul DNA
  • Same thermocycler protocol as 8.1.2007

• Gel
  • Ran 1% gel for 60 minutes at 100V with leucine zipper samples (5ul sample with 2ul of loading dye)
  • Ran 2% gel for 60 minutes at 100V with IgAbc samples (5ul sample with 2 ul of loading dye)
  • Ran gel too long, weird results

• Spec
  • IgAbc F: ~400 ng/ul
  • IgAbc R: ~400 ng/ul
  • Fos: ~400 ng/ul
  • JunB: ~400 ng/ul
  • GCN4: ~400 ng/ul (after PCR purification ~100 ng/ul)

Conserv IgAbc F, Conserv IgAbc R, log-2 ladder, Fos, JunB, GCN4

• PCR Purification
  • Used MinElute columns to purify PCR product above
  • Eluted in 10ul of water

• PCR
  • Liberal IgAbc: 40ul PCR Supermix, .4ul IgAb-F and IgAb-R, 5ul gel purification DNA

• Gel
  • Ran 1% gel for 30 minutes at 100V with samples that didn't work before (5ul sample with 2ul of loading dye)
  • Looks like leucine zippers worked
  • Looks like Conserv IgAbc F did not work
  • Looks like Conserv IgAbc R might no have worked

• Digest
  • Cut all samples from PCR purification and signal sequence (tube A from 8.1.2007)
  • Template (20ul rxns): 3ul DNA, 2ul NEB2, .2ul BSA, .5ul EcoR1, .5ul Pst1, rest water
  • Protocol: 1hr@37C, 20mins@80C

• Ligation
  • Template: .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
  • Ligated all digested samples except IgAbc-F
  • 25mins@roomtemp,10mins@65C

IgAbc F ligation, IgAbc R ligation, IgAbc complete (no muts), liberal IgAbc PCR, IgAbc F PCR, IgAbc R PCR

• Gel
  • 1% for 30 minutes at 100V to diagnose problem with the conservative ligations
  • Looks like something went wrong with B + C ligation reaction (IgAbc F), but not with C + D ligation (IgAbc R)
  • Liberal approach did not produce the correct result

• 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 Supermix, .4ul of each primer, 1ul of DNA
  • Replicated existing PCR product of the following samples:
    • B (Part 1 of IgAbc)
    • C (Part 2 of IgAbc)
    • D (Part 3 of IgAbc)
    • E (Complete IgAbc without mutated Pst1 sites)
  • Protocol: Same thermocycler protocol as 8.1.2007