User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/02
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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
- Template (20ul rxns): 3ul DNA (of each), 2ul NEB4, .5ul Sap1, rest water
- 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
- Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation product
- 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
- 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