Friday, August 19th
Tasks:
Michelle
- Ran gel on Cas9 linearized for signal sequences
- Gel extracted the upper crest
- Pt 1: 0.2561 g (dissolved in 0.85 mL GEX)
- Pt 2: 0.3051 g (dissolved in 1.00 mL GEX)
- Nanodrop: 31.4 ng/uL, 260/280: 1.68, 260/230: 1.17
- PCR for GFP 1 to test template DNA amounts
- Made a 1:10 dilution of the 42.5 ng/uL miniprepped stock of template
- Reactions:
- 1.0 uL of template reaction
- 1 uL of 4.25 ng/uL GFP template
- 1 uL of 10 uM forward primer
- 1 uL of 10 uM reverse primer
- 1 uL of DMSO
- 21 uL of nfH2O
- 25 uL of 2X OneTaq Master Mix
- 0.5 uL of template reaction
- 0.5 uL of 4.25 ng/uL GFP template
- 1 uL of 10 uM forward primer
- 1 uL of 10 uM reverse primer
- 1 uL of DMSO
- 21.5 uL of nfH2O
- 25 uL of 2X OneTaq Master Mix
- Conditions: 95°C (5:00) | 95°C (0:07), 54°C (0:10), 72°C (0:43) | 72°C (5:00)
- Ran gel of GFP test PCR
- Used NEB Purple Loading Dye 6X
- 2 uL 2 log purple ladder + 6 uL dye
- 50 uL PCR reaction + 10 uL dye
- 15 uL of reaction into each well
- Results were the same as previous PCRs, did not extract
Paul
- Did the presentation
- Looked over Cas9 BB insert primers
- Looked at GFP primers
Sam
- Troubleshot GFP PCR. It’s making 500 bp bands instead of 800, so I tried to find other binding sites for the primers. Couldn’t find anything
- Started work on Resisting Resistance public kit
Tasfia
- Re-ran PCR/gel for GFP/mCherry for GG, but only for GFP
- Running with an annealing temperature of 54°C instead of 51°C (which is what we were doing before)
- NEB recommends an annealing temperature of 53°C, but we ran at 54°C as per Bradley’s suggestion at this morning’s meeting
- PCR protocol is otherwise exactly the same as the one used the day before (18 August 2016)
- Ran a gel of GFPs
- Out of 60 μL PCR product + dye per tube, put 20 μL in each well
- Did not extract
- Made progress on “Attributions” web content
- Made progress on “Project Overview” web content
Michelle
- Ran gel on Cas9 linearized for signal sequences
- Gel extracted the upper crest
- Pt 1: 0.2561 g (dissolved in 0.85 mL GEX)
- Pt 2: 0.3051 g (dissolved in 1.00 mL GEX)
- Nanodrop: 31.4 ng/uL, 260/280: 1.68, 260/230: 1.17
- PCR for GFP 1 to test template DNA amounts
- Made a 1:10 dilution of the 42.5 ng/uL miniprepped stock of template
- Reactions:
- 1.0 uL of template reaction
- 1 uL of 4.25 ng/uL GFP template
- 1 uL of 10 uM forward primer
- 1 uL of 10 uM reverse primer
- 1 uL of DMSO
- 21 uL of nfH2O
- 25 uL of 2X OneTaq Master Mix
- 0.5 uL of template reaction
- 0.5 uL of 4.25 ng/uL GFP template
- 1 uL of 10 uM forward primer
- 1 uL of 10 uM reverse primer
- 1 uL of DMSO
- 21.5 uL of nfH2O
- 25 uL of 2X OneTaq Master Mix
- Conditions: 95°C (5:00) | 95°C (0:07), 54°C (0:10), 72°C (0:43) | 72°C (5:00)
- 1.0 uL of template reaction
- Ran gel of GFP test PCR
- Used NEB Purple Loading Dye 6X
- 2 uL 2 log purple ladder + 6 uL dye
- 50 uL PCR reaction + 10 uL dye
- 15 uL of reaction into each well
- Results were the same as previous PCRs, did not extract
Paul
- Did the presentation
- Looked over Cas9 BB insert primers
- Looked at GFP primers
Sam
- Troubleshot GFP PCR. It’s making 500 bp bands instead of 800, so I tried to find other binding sites for the primers. Couldn’t find anything
- Started work on Resisting Resistance public kit
Tasfia
- Re-ran PCR/gel for GFP/mCherry for GG, but only for GFP
- Running with an annealing temperature of 54°C instead of 51°C (which is what we were doing before)
- NEB recommends an annealing temperature of 53°C, but we ran at 54°C as per Bradley’s suggestion at this morning’s meeting
- PCR protocol is otherwise exactly the same as the one used the day before (18 August 2016)
- Running with an annealing temperature of 54°C instead of 51°C (which is what we were doing before)
- Ran a gel of GFPs
- Out of 60 μL PCR product + dye per tube, put 20 μL in each well
- Did not extract
- Made progress on “Attributions” web content
- Made progress on “Project Overview” web content
