User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/24

Cellular Adhesion

 * Colony PCR
 * Template: 20ul Supermix, .8ul VF2 and VR, 1 ul of cell dilution (colony in 100ul of water)
 * Picked three colonies from each plated sample to PCR (five tags and GCN4)
 * Picked a large, medium, and small colony (in that order)
 * Some plates only had three colonies


 * Liquid Cultures
 * Inoculated 8ml Amp/Cl LB with antibody tags and GCN4 from yesterday's plate colonies
 * Grew up in 37C overnight


 * Gel
 * Ran 1% gel for 30 minutes at 95V with samples (5ul sample with 2ul of loading dye)
 * Again, poor resolution with 1% gel, but relative lengths look good


 * PCR Purification
 * Used MinElute columns to purify the NEW B, NEW C, NEW D from 1.23 PCR
 * Eluted in 10ul of water


 * Digest
 * Template for IGA (single): 3ul DNA (each), 2ul NEB2, .5ul Sap1, rest water (20ul rxn)
 * NEW B + NEW C
 * NEW C + NEW D
 * NEW B + NEW C + NEW D
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Ligation
 * 6ul of IGA digest DNA, 7ul water, 1.5ul ligase buffer, .5ul ligase
 * Protocol: 30mins@roomtemp,10mins@65C