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

log-2 ladder, 6His 1, 6His 2, 6His 3, FLAG 1, FLAG 2, FLAG 3, Myc 1, Myc 2, Myc 3, HA 1, HA 2, NEW GCN4 1, NEW GCN4 2, NEW GCN4 3

• 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

log-2 ladder, B + C with D, C + D with D, B + C + D

• 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