User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/18

Cellular Adhesion

 * Solid Culture
 * No colonies -> I guess I should have have PCR purified the samples
 * Actually samples were grown up on Cl/Kan and did not have Kan resistance -> no cells


 * PCR Purification
 * Used MinElute columns to PCR purify autotransporter (4), antibody tags(4), and GCN4 leucine zipper (1)
 * Eluted in 10ul of water


 * Digest
 * Template: 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
 * B+C with D (IgA primers)
 * C+D with B (IgA primers)
 * B+C with D (BB primers)
 * C+D with B (BB primers)
 * 6His Tag
 * FLAG Tag
 * Myc Tag
 * HA Tag
 * GCN4 Leucine Zipper
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Ligation
 * Template: .2ul vector (1AC3 for antibody tags and leucine zipper, 1AK3 for autotransporter), 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
 * Ligated all samples from today's digest
 * 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 Cl or Kan (made some more Cl/Amp and Kan/Amp plates) and grew up at 37C overnight