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

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

• 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

• Oligos
  • Mut2_Pst1b-F
  • Mut2_Pst1b-R
  • Resuspended at 50uM

• PCR
  • Template: 40ul PCR Supermix, .4ul of each primer, and .4ul N. gonorrhea DNA
    • 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
    • E: Full IgA beta-core IgAb-F and IgAb-R
    • Same protocol as 8.1.2007

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

• 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)