User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/25

Cellular Adhesion

 * Gel
 * Ran 1% gel for 25 minutes at 95V with samples (5ul sample with 2ul of loading dye)
 * Looks good, HA 2 has unusual second band


 * Glycerol
 * The third colony of 6His, FLAG, Myc, and HA did not grow -> threw away
 * Took 1.6ml of remaining liquid culture and created 10% glycerol stocks


 * Miniprep
 * Extracted DNA from remaining liquid culture (~6ml)
 * Eluted in 50ul water
 * Used QIAquick columns instead of QIAprep columns and it looks like it effected the DNA yield (~30ng/ul)


 * Sequencing
 * Template: 3ul Miniprep DNA, .3ul primer, 6.7ul water
 * Probably won't work due to insufficient DNA
 * 1.25.2008


 * Gel
 * Ran 1% gel for 25 minutes at 95V with samples (5ul sample with 2ul of loading dye)
 * Looks good, although B + C + D might not be correct


 * PCR Purification
 * Used MinElute columns
 * Eluted in 10ul of water


 * Digest
 * Template for IGA: 3ul DNA (each), 2ul NEB2, .5ul Sap1, rest water (20ul rxn)
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Ligation
 * 6ul of IGA digest DNA, 7ul water, 1.5ul ligase buffer, .5ul ligase
 * Protocol: 30mins@roomtemp,10mins@65C


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation
 * Used samples from ligation above
 * Same protocol as 1.21.2008