User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/09

Cellular Adhesion

 * Oligos
 * IgAbfull-F
 * Resuspended at 50uM


 * Colony PCR
 * Performed on seven colonies of 8.3.2007 IgAbc-R plate
 * Template: 20ul Supermix, .8ul VF2 and VR, 1 ul of cell dilution (colony in 100ul of water)
 * Plated picked colonies on Amp/Cl plate and grew up at 37C all day and overnight
 * Note: Annealing temp was initially 45C, noticed after 5 cycles, restarted protocol without initial 95C denature and correct annealing temperature
 * Protocol:
 * 95C for 15 mins
 * 94C for 30 secs
 * 55C for 30 secs
 * 68C for 1.5 mins
 * REPEAT 2-4 39 times
 * 68C for 10 mins
 * 4C FOREVER


 * PCR
 * Full Iga beta: IgAbfull-F and IgAb-R
 * Template: 40ul PCR Supermix, .4ul of each primer, and .4ul N. gonorrhea DNA
 * Same protocol as the colony PCR


 * Digest
 * Template (20ul rxns): 3ul DNA (of each), 2ul NEB4, .5ul Sap1, rest water
 * C + D with B
 * B + C
 * C + D
 * Thermocycler protocol: 1hr@37C, 20mins@65C


 * Gel
 * Ran 1.5% for 50 minutes at 100V to see if PCR product is correct length


 * Liquid Culture
 * Grew up cultures in 8ml Amp/Cl LB
 * GCN4 #1 from 8.4 colony PCR backup plate
 * GCN4 #3 from 8.4 colony PCR backup plate
 * IgAbc #8 from 8.9 colony PCR cell dilution


 * Ligation
 * Performed on today's digest tubes
 * Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
 * Thermocycler protocol: 1hr@16C,10mins@65C