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
- 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