User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/17
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Cellular Adhesion
- Gel
- Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
- Repeat of last gel yesterday, but with more fragments of known length
- Same conclusion as last gel of 8.16.2007
- PCR Purification
- Used MinElute columns to PCR purify tubes B+C, C+D
- Eluted in 10ul of water
- Digest
- Template (20ul rxns): 3ul DNA, 2ul NEB2, .2ul BSA, .5ul EcoR1, .5ul Pst1, rest water
- 6His Tag
- FLAG Tag
- Myc Tag
- HA Tag
- GCN4 Leucine Zipper
- Thermocycler protocol: 1hr@37C, 20mins@80C
- Note: DNA was not PCR purified (it shouldn't matter)
- Template (20ul rxns): 3ul DNA, 2ul NEB2, .2ul BSA, .5ul EcoR1, .5ul Pst1, rest water
- PCR
- Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of PCR product
- B: Part 1 of IgA beta-core IgAb-F and Mut_Pst1a-R
- C: Part 2 of IgA beta-core Mut_Pst1a-F and Mut2_Pst1b-R
- D: Part 3 of IgA beta-core Mut2_Pst1b-F and IgAb-R
- Protocol same as 8.1.2007
- Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of PCR product
- Digest
- Template: 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
- B+C with D
- C+D with B
- Thermocycler protocol: 1hr@37C, 20mins@80C
- Template: 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
- Ligation
- Template: .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
- Ligated all digested samples from the first digest of the day (antibody tags and GCN4 leucine zipper)
- Thermocycler protocol: 30mins@roomtemp,10mins@65C
- Ligation
- Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
- Ligated all digested samples from the second digest of the day (IgAbc samples)
- Thermocycler protocol: 30mins@roomtemp,10mins@65C
- Transformation
- 50ul of TOP10 cells on ice with 2ul of DNA for 30mins
- 45secs heat shock at 42C using water bath
- Added 300ul of SOC
- Incubated for 1hr at 37C
- Plated on Amp/Cl and grew up at 37C overnight
- PCR
- Template: 40ul PCR Supermix, .4ul of each IgA primer (.8ul of each BB primer), and 1ul of ligation product
- B+C with D
- C+D with B
- Both samples PCRed with both IgA primers and BB primers
- Protocol same as 8.1.2007
- Template: 40ul PCR Supermix, .4ul of each IgA primer (.8ul of each BB primer), and 1ul of ligation product
- Gel
- Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
- BANDS ARE AT THE CORRECT LENGTH!!! WE HAVE COMPLETE IGA BETA-CORE WITH MUTATIONS!!!