User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/17

Cellular Adhesion

log2-ladder, B, C, D, B + C, C + D, B + C + D (IgA primers), B + C + D (BB primers), Complete IgAbc without muts, log-2 ladder

• Gel
  • Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
  • Repeat of last gel yesterday, but with more fragments of known length
  • Same conclusion as last gel of 8.16.2007

• PCR Purification
  • Used MinElute columns to PCR purify tubes B+C, C+D
  • Eluted in 10ul of water

• Digest
  • Template (20ul rxns): 3ul DNA, 2ul NEB2, .2ul BSA, .5ul EcoR1, .5ul Pst1, rest water
    • 6His Tag
    • FLAG Tag
    • Myc Tag
    • HA Tag
    • GCN4 Leucine Zipper
  • Thermocycler protocol: 1hr@37C, 20mins@80C
  • Note: DNA was not PCR purified (it shouldn't matter)

• PCR
  • Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of PCR product
    • B: Part 1 of IgA beta-core IgAb-F and Mut_Pst1a-R
    • C: Part 2 of IgA beta-core Mut_Pst1a-F and Mut2_Pst1b-R
    • D: Part 3 of IgA beta-core Mut2_Pst1b-F and IgAb-R
  • Protocol same as 8.1.2007

• Digest
  • Template: 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
    • B+C with D
    • C+D with B
  • Thermocycler 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 from the first digest of the day (antibody tags and GCN4 leucine zipper)
  • Thermocycler protocol: 30mins@roomtemp,10mins@65C

• Ligation
  • Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
  • Ligated all digested samples from the second digest of the day (IgAbc samples)
  • Thermocycler 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 IgA primer (.8ul of each BB primer), and 1ul of ligation product
    • B+C with D
    • C+D with B
    • Both samples PCRed with both IgA primers and BB primers
  • Protocol same as 8.1.2007

B+C with D (IgA primers), B+C with D (BB primers), C+D with B (IgA primers), C+D with B (BB primers), log-2 ladder

• Gel
  • Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
  • BANDS ARE AT THE CORRECT LENGTH!!! WE HAVE COMPLETE IGA BETA-CORE WITH MUTATIONS!!!