User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/15

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Cellular Adhesion

  • PCR Purification
    • Used MinElute columns to PCR purify tube D
    • Eluted in 10ul of water
  • Digest
    • B + C with D: 3ul D DNA, 3ul B + C DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
    • Thermocycler protocol: 1hr@37C, 20mins@80C
  • Ligation
    • Performed on digest tube 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 1ul of ligation product
      • B + C with D: IgAb-F and IgAb-R
      • B + C with D: BB_f and BB_r (.8ul of each)
    • Same thermocycler protocol as 8.1.2007
B+C with D (IgA primers), B+C with D (BB primers), PCR D (from yesterday), Complete IgAbc without muts, log-2 ladder
B+C with D (IgA primers), B+C with D (BB primers), PCR D (from yesterday), 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)
    • Didn't work, but new primers are here anyway
Tube B, Tube C, Tube D, Tube E, log-2 ladder
Tube B, Tube C, Tube D, Tube E, log-2 ladder
  • Gel
    • Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
    • Everything looks good, proceeding to put the pieces together
  • PCR Purification
    • Used MinElute columns to PCR purify tubes B, C, and D
    • Eluted in 10ul of water
  • Digest
    • B + C + D: 3ul DNA (each), 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
    • Thermocycler protocol: 1hr@37C, 20mins@80C
  • Spec
    • B: 67.5 ng/ul
    • C: 54 ng/ul
    • D: 100.2 ng/ul
  • Ligation
    • Performed on digest tube 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 1ul of ligation product
      • B + C + D: IgAb-F and IgAb-R
      • B + C + D: BB_f and BB_r (.8ul of each)
    • Same thermocycler protocol as 8.1.2007
B + C + D (with IgA primers), B + C + D (with BB primers), Complete IgAbc without muts, log-2 ladder
B + C + D (with IgA primers), B + C + D (with 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)
    • It didn't work, what a surprise
  • PCR Purification
    • Used MinElute columns to PCR purify tubes B, C, and D
    • Eluted in 10ul of water
    • Messed up and eluted in catch tube, definitely contaminated DNA with some PE buffer
    • Used for digest, threw away the rest of the DNA
  • Digest
    • Template (20ul rxn): 3ul B + C + D DNA, 2ul NEB2, .2ul BSA, .5ul EcoR1, .5ul Pst1, rest water
    • Done for both the PCRs (one using IgA primers, the other using BB primers)
    • Protocol: 1hr@37C, 20mins@80C
    • PCR didn't amplify correct length band, threw digest away
  • PCR
    • Poked the gel where the correct length DNA segment should be and dipped in Supermix for template
    • Template: 40ul PCR Supermix, and .4ul of each primer
      • B + C + D: IgAb-F and IgAb-R
      • B + C + D: BB_f and BB_r (.8ul of each)
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