User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/13

Cellular Adhesion

 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * Something is going wrong between first ligation and second ligation


 * Digest
 * Template: 3ul DNA, 2ul NEB2, .2ul BSA, .5ul of EcoRI and Pst1, rest water (20ul)
 * IgAbc F (B + C with D)
 * IgAbc R (C + D with B)
 * PCR didn't work, threw away


 * Antibody Tag Primers
 * Ordered (more info later)


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (20ul sample with 5ul of loading dye)
 * Cut band at about 700bp of B + C for gel purification


 * Gel Purification
 * QIAEX gel purification
 * Eluted in 20ul of water


 * Spec
 * B + C: 82.5 ng/ul


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, 1ul gel purification product
 * B + C: Run with normal primers (IgAb-F and Mut_Pst1b-R)
 * B + C diagnostic: Run with normal primers plus Mut_Pst1a-R


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * Looks like the diagnostic PCR has a band 100bp shorter than B+C
 * Looks promising
 * What is creating this band at 200bp?


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


 * Ligation
 * Performed on digest tubes above
 * Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
 * Thermocycler protocol: 30mins@roomtemp,10mins@65C


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and 2ul of ligation product
 * B + C with D: IgAb-F and IgAb-R
 * Same thermocycler protocol as 8.1.2007