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

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Cellular Adhesion

B + C with D, C + D with D, Complete IgAbc without muts, log-2 ladder
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
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
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
    • Same thermocycler protocol as 8.1.2007
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