User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/12

Cellular Adhesion

 * Glycerol
 * Took 1.6ml of liquid culture and created 10% glycerol stocks
 * IgAss + Fos #1
 * IgAss + Fos #2
 * IgAss + Fos #3
 * IgAss + JunB #1
 * IgAss + JunB #2
 * IgAss + JunB #3
 * GCN4 #4
 * GCN4 #5
 * GCN4 #6


 * Miniprep
 * Extracted DNA from remaining liquid culture (~6ml)
 * Eluted in 50ul water


 * Digest
 * Template (20ul rxns): 3ul DNA, 2ul NEB4, .5ul Sap1, rest water
 * B: Part 1 of IgAbc
 * C: Part 2 of IgAbc
 * D: Part 3 of IgAbc
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Ligation
 * Performed on today's digest tubes
 * Template (50ul rxns): 10ul of each digest, 5ul ligase buffer, .5ul ligase, rest water
 * B + C
 * C + D
 * Thermocycler protocol: 1hr@16C,15mins@65C


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation product
 * 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


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * Everything looks fine


 * Digest
 * B + C with D: 1.5ul D digest, 1.5ul B + C DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
 * C + D with B: 1.5ul B digest, 1.5ul C + D DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Ligation
 * Template (50ul rxns): 20ul of each digest, 8.5ul of remaining digested DNA(B and D), 5ul ligase buffer, .5ul ligase, rest water
 * B + C with D
 * C + D with B
 * Thermocycler protocol: 1hr@16C,10mins@65C


 * PCR
 * Conservative IgAbc F (B + C with D): 40ul PCR Supermix, .4ul IgAb-F and IgAb-R, 1ul ligation product
 * Conservative IgAbc R (C + D with B): 40ul PCR Supermix, .4ul IgAb-F and IgAb-R, 1ul ligation product
 * Same thermocycler protocol as 8.1.2007