User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/14

Cellular Adhesion

 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (20ul sample with 5ul of loading dye)
 * There is no band at the correct length
 * Problem might be caused by PCR: primer is now binding to entire piece of DNA instead of just half of it


 * PCR
 * Template: 20ul PCR Supermix, .2ul VF2 and VR, .5ul miniprep DNA
 * SS+Fos #1
 * SS+Fos #2
 * SS+Fos #3
 * SS+JunB #1
 * SS+JunB #2
 * SS+JunB #3
 * Protocol: Same as 8.1.2007


 * Gradient PCR
 * Template: 10ul PCR Supermix, .2ul BB_f and BB_r, .5ul ligation DNA (B+C with D)
 * Ran eight samples from 55-70C on gradient PCR (other than annealing temp, everything else is the same as 8.1.2007 protocol)
 * Template: 10ul PCR Supermix, .1ul IgAb-F and IgAb-R, .5ul ligation DNA (B+C with D)
 * Ran eight samples from 55-70C on gradient PCR (other than annealing temp, everything else is the same as 8.1.2007 protocol)


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (10ul sample with 4ul of loading dye)
 * Looks like everything worked except SS+JunB #2 (~300bp)


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (10ul sample with 4ul of loading dye)
 * All bands are at 300bp
 * WHAT IS THIS?!?!
 * Possible Solution: By not PCR purifying tube D, it amplified D instead of the entire fragment


 * PCR
 * Template: 40ul PCR Supermix, .4ul VF2 and VR, .8ul DNA
 * PCRed tube D to make some more
 * Protocol: Same as 8.1.2007