User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/04


 * PCR Purification
 * Used MinElute columns to purify B, C, D, and IgAbc complete (no muts) PCR product from yesterday
 * Eluted in 10ul of water


 * 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 (Signal Sequence, IgAbc R, Fos, JunB, GCN4)
 * Plated picked colonies on Amp/Cl plate and grew up at 37C all day and overnight
 * Protocol:
 * 95C for 15 mins
 * 94C for 30 secs
 * 55C for 30 secs
 * 68C for 1 min
 * REPEAT 2-4 39 times
 * 68C for 10 mins
 * 4C FOREVER


 * Gel
 * Ran 1% for 30 minutes at 100V to ensure that PCR worked
 * Everything looks good


 * Gel
 * Ran 1.5% for 50 minutes at 100V to see if PCR product is correct length
 * Expected sizes (without VF and VR2 additions): Signal Sequence (140 bp), IgAbc (892 bp), Fos (183 bp), JunB (180 bp), GCN4 (156 bp)
 * Looks like we might have some correct inserts
 * Onwards to sequencing


 * Liquid Cultures
 * Grew up samples in 8ml of Amp/Cl media for sequencing overnight in 37C incubator (inoculated with colony PCR cell dilution):
 * Signal Sequence #2
 * Signal Sequence #3
 * IgAbc #1
 * Fos #1
 * Fos #3
 * JunB #3
 * GCN4 #2