User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/03

Cellular Adhesion

 * Miniprep
 * QIAGEN miniprep on liquid cultures from yesterday (leucine zippers)
 * Spun down for 10min at 3000rcf then followed protocol


 * Spec
 * DNA containing Fos: 51.8 ng/ul
 * DNA containing JunB: 51.4 ng/ul
 * DNA containing GCN4: 90.0 ng/ul


 * 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
 * DNA containing Fos: 40ul PCR Supermix, .4ul Fos-F and Fos-R, 2ul DNA
 * DNA containing JunB: 40ul PCR Supermix, .4ul JunB-F and JunB-R, 2ul DNA
 * DNA containing GCN4: 40ul PCR Supermix, .4ul GCN4-F and GCN4-R, 1ul DNA
 * Same thermocycler protocol as 8.1.2007


 * Gel
 * Ran 1% gel for 60 minutes at 100V with leucine zipper samples (5ul sample with 2ul of loading dye)
 * Ran 2% gel for 60 minutes at 100V with IgAbc samples (5ul sample with 2 ul of loading dye)
 * Ran gel too long, weird results


 * Spec
 * IgAbc F: ~400 ng/ul
 * IgAbc R: ~400 ng/ul
 * Fos: ~400 ng/ul
 * JunB: ~400 ng/ul
 * GCN4: ~400 ng/ul (after PCR purification ~100 ng/ul)


 * PCR Purification
 * Used MinElute columns to purify PCR product above
 * Eluted in 10ul of water


 * PCR
 * Liberal IgAbc: 40ul PCR Supermix, .4ul IgAb-F and IgAb-R, 5ul gel purification DNA


 * Gel
 * Ran 1% gel for 30 minutes at 100V with samples that didn't work before (5ul sample with 2ul of loading dye)
 * Looks like leucine zippers worked
 * Looks like Conserv IgAbc F did not work
 * Looks like Conserv IgAbc R might no have worked


 * Digest
 * Cut all samples from PCR purification and signal sequence (tube A from 8.1.2007)
 * Template (20ul rxns): 3ul DNA, 2ul NEB2, .2ul BSA, .5ul EcoR1, .5ul Pst1, rest water
 * 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 except IgAbc-F
 * 25mins@roomtemp,10mins@65C


 * Gel
 * 1% for 30 minutes at 100V to diagnose problem with the conservative ligations
 * Looks like something went wrong with B + C ligation reaction (IgAbc F), but not with C + D ligation (IgAbc R)
 * Liberal approach did not produce the correct result


 * 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 Supermix, .4ul of each primer, 1ul of DNA
 * Replicated existing PCR product of the following samples:
 * B (Part 1 of IgAbc)
 * C (Part 2 of IgAbc)
 * D (Part 3 of IgAbc)
 * E (Complete IgAbc without mutated Pst1 sites)
 * Protocol: Same thermocycler protocol as 8.1.2007