User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/02

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


 * 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


 * 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