User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/13

Cellular Adhesion

B + C with D, C + D with D, 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)
  • Something is going wrong between first ligation and second ligation

• Digest
  • Template: 3ul DNA, 2ul NEB2, .2ul BSA, .5ul of EcoRI and Pst1, rest water (20ul)
    • IgAbc F (B + C with D)
    • IgAbc R (C + D with B)
  • PCR didn't work, threw away

• Antibody Tag Primers
  • Ordered (more info later)

B+C PCR, log-2 ladder

• Gel
  • Ran 1.5% gel for 30 minutes at 100V with samples (20ul sample with 5ul of loading dye)
  • Cut band at about 700bp of B + C for gel purification

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

• Spec
  • B + C: 82.5 ng/ul

• PCR
  • Template: 40ul PCR Supermix, .4ul of each primer, 1ul gel purification product
  • B + C: Run with normal primers (IgAb-F and Mut_Pst1b-R)
  • B + C diagnostic: Run with normal primers plus Mut_Pst1a-R

B+C, B+C diagnostic, log-2 ladder

• Gel
  • Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
  • Looks like the diagnostic PCR has a band 100bp shorter than B+C
  • Looks promising
  • What is creating this band at 200bp?

• Digest
  • Template (20ul rxns): 3ul DNA (of each), 2ul NEB4, .5ul Sap1, rest water
    • B + C with 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: 30mins@roomtemp,10mins@65C

• PCR
  • Template: 40ul PCR Supermix, .4ul of each primer, and 2ul of ligation product
    • B + C with D: IgAb-F and IgAb-R
  • Same thermocycler protocol as 8.1.2007