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

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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
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
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
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