User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/23

Cellular Adhesion

 * Gel
 * Ran 1% gel for 25 minutes at 95V with samples (5ul sample with 2ul of loading dye)
 * Not a great gel, but the smudges around 100 bp look promising
 * Extra band in GCN4 sample


 * PCR Purification
 * Used MinElute columns to purify the five tags and one zipper from 1.22 PCR
 * Eluted in 10ul of water


 * Digest
 * Ran on PCR purification above
 * Template for tags: 3ul DNA (each), 2ul NEB2, .5ul EcoR1, .5ul Pst1, rest water (20ul rxn)
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Ligation
 * Ran on digest samples above
 * Template for assembly: .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
 * 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 primer, and .4ul of of N. gonorrhea DNA
 * NEW B: Part 1 of IgA beta-core IgAb-F and NEW_Mut_Pst1a-R
 * NEW C: Part 2 of IgA beta-core NEW_Mut_Pst1a-F and NEW_Mut2_Pst1b-R
 * NEW D: Part 3 of IgA beta-core NEW_Mut2_Pst1b-F and IgAb-R
 * Same protocol as 1.21.2008


 * Solid Culture
 * Grew up samples from transformation on Amp/Cl plates overnight at 37C