User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/24: Difference between revisions
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*** NEW B + NEW C + NEW D | *** NEW B + NEW C + NEW D | ||
** Thermocycler protocol: 1hr@37C, 20mins@80C | ** 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 |
Latest revision as of 13:44, 24 January 2008
Cellular Adhesion
- Colony PCR
- Template: 20ul Supermix, .8ul VF2 and VR, 1 ul of cell dilution (colony in 100ul of water)
- Picked three colonies from each plated sample to PCR (five tags and GCN4)
- Picked a large, medium, and small colony (in that order)
- Some plates only had three colonies
- Liquid Cultures
- Inoculated 8ml Amp/Cl LB with antibody tags and GCN4 from yesterday's plate colonies
- Grew up in 37C overnight
- Gel
- Ran 1% gel for 30 minutes at 95V with samples (5ul sample with 2ul of loading dye)
- Again, poor resolution with 1% gel, but relative lengths look good
- PCR Purification
- Used MinElute columns to purify the NEW B, NEW C, NEW D from 1.23 PCR
- Eluted in 10ul of water
- Digest
- Template for IGA (single): 3ul DNA (each), 2ul NEB2, .5ul Sap1, rest water (20ul rxn)
- NEW B + NEW C
- NEW C + NEW D
- NEW B + NEW C + NEW D
- Thermocycler protocol: 1hr@37C, 20mins@80C
- Template for IGA (single): 3ul DNA (each), 2ul NEB2, .5ul Sap1, rest water (20ul rxn)
- Ligation
- 6ul of IGA digest DNA, 7ul water, 1.5ul ligase buffer, .5ul ligase
- Protocol: 30mins@roomtemp,10mins@65C