User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/19

Cellular Adhesion

 * Liquid Culture
 * Inoculated 8ml Amp/Cl LB with antibody tags and GCN4 from plate colonies
 * Inoculated 8ml Amp/Kan LB with autotransporter from plate colonies
 * Put in incubator at 12:15 AM


 * 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
 * B+C w/ D (IgA primers)
 * C+D w/ B (IgA primers)
 * B+C w/ D (BB primers)
 * C+D w/ B (BB primers)
 * 6His Tag
 * FLAG Tag
 * Myc Tag
 * HA Tag
 * GCN4 Leucine Zipper
 * Grew up liquid cultures of all samples for miniprepping (if gel shows correct insert)
 * Protocol:
 * 95C for 15 mins
 * 94C for 30 secs
 * 55C for 30 secs
 * 68C for 1.5 min
 * REPEAT 2-4 39 times
 * 68C for 10 mins
 * 4C FOREVER


 * Gel
 * Ran 1.5% for 30 minutes at 100V to see if PCR product is correct length (5ul of PCR product with 2ul of dye)
 * B+C w/ D (IgA primers): 2 unique (none look right)
 * C+D w/ B (IgA primers): 2 unique (none look right)
 * B+C w/ D (BB primers): 2 unique (none look right, contains longest fragment of all autotransporters)
 * C+D w/ B (BB primers): 2 unique (none look right)
 * 6His Tag: 3 unique (sequence all)
 * FLAG Tag: 3 unique (sequence all)
 * Myc Tag: 2 unique (sequence both)
 * HA Tag: 2 unique (sequence both)
 * GCN4 Leucine Zipper: 1 unique (sequence)