User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/17

Cellular Adhesion



 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * Repeat of last gel yesterday, but with more fragments of known length
 * Same conclusion as last gel of 8.16.2007


 * PCR Purification
 * Used MinElute columns to PCR purify tubes B+C, C+D
 * Eluted in 10ul of water


 * Digest
 * Template (20ul rxns): 3ul DNA, 2ul NEB2, .2ul BSA, .5ul EcoR1, .5ul Pst1, rest water
 * 6His Tag
 * FLAG Tag
 * Myc Tag
 * HA Tag
 * GCN4 Leucine Zipper
 * Thermocycler protocol: 1hr@37C, 20mins@80C
 * Note: DNA was not PCR purified (it shouldn't matter)


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of PCR product
 * 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
 * Protocol same as 8.1.2007


 * Digest
 * Template: 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
 * B+C with D
 * C+D with B
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Ligation
 * Template: .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
 * Ligated all digested samples from the first digest of the day (antibody tags and GCN4 leucine zipper)
 * Thermocycler protocol: 30mins@roomtemp,10mins@65C


 * Ligation
 * Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
 * Ligated all digested samples from the second digest of the day (IgAbc samples)
 * Thermocycler protocol: 30mins@roomtemp,10mins@65C


 * Transformation
 * 50ul of TOP10 cells on ice with 2ul of DNA for 30mins
 * 45secs heat shock at 42C using water bath
 * Added 300ul of SOC
 * Incubated for 1hr at 37C
 * Plated on Amp/Cl and grew up at 37C overnight


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each IgA primer (.8ul of each BB primer), and 1ul of ligation product
 * B+C with D
 * C+D with B
 * Both samples PCRed with both IgA primers and BB primers
 * Protocol same as 8.1.2007


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * BANDS ARE AT THE CORRECT LENGTH!!! WE HAVE COMPLETE IGA BETA-CORE WITH MUTATIONS!!!