User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/02

Cellular Adhesion

• PCR Purification
  • Used MinElute columns to PCR purify the six samples from yesterday (A-F)
  • Eluted in 10ul of water

• Spec
  • B(IgA-bc Part 1): 104.5 ng/ul
  • C(IgA-bc Part 2): 76.3 ng/ul
  • D(IgA-bc Part 3): 87.1 ng/ul

• Digest
  • Template (20ul rxns): 3ul DNA (of each), 2ul NEB4, .5ul Sap1, rest water
    • B + C + D
    • B + C
    • C + D
  • Thermocycler protocol: 1hr@37C, 20mins@65C

• Ligation
  • Performed on digest tubes above
  • Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
  • Thermocycler protocol: 1hr@16C,10mins@65C

• PCR
  • Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation product
    • B + C + D with IgAb-F and IgAb-R
    • B + C with IgAb-F and Mut_Pst1b-R
    • C + D with Mut_Pst1a-F and IgAb-R
  • Same thermocycler protocol as 8.1.2007

• Liquid Cultures
  • Inoculated 8ml of LB Tet with Fos containing plasmid
  • Inoculated 8ml of LB Tet with JunB containing plasmid
  • Inoculated 8ml of LB Kan with BBa_C2001
  • Grew up overnight at 37C

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

B + C + D (892 bp), B + C + D (892 bp), log-2 ladder

• Gel
  • Ran 1% gel for 40 minutes at 100V with samples (5ul sample with 2ul of loading dye)
  • Bands might be in the right place (a little short, but hard to tell)
  • Cut and purified band that was near the correct region

• Spec
  • B + C: 192 ng/ul
  • C + D: 218 ng/ul

• Digest
  • B + C with D: 3ul D DNA, 1.5ul B + C DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
  • C + D with B: 3ul B DNA, 1.5ul C + D DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
  • Thermocycler protocol: 1hr@37C, 20mins@65C

• Gel Purification
  • QIAEX gel purification
  • Eluted in 20ul of water

• Ligation
  • Performed on digest tubes above
  • Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
  • Thermocycler protocol: overnight@16C,10mins@65C