Tuesday, August 30th
Tasks:
Jordan
- Started cytosolic GFP cultures for tomorrow
- Interviewed Dr. Postelnick
- Worked on travel grant
- Read instructions for millipore filters
Michelle
- Ran western blot
- We successfully expressed Cas9
- There are no bands in lanes 2-4 because Thush didn’t induce the promoter but Ben offered to run another Western after inducing
- It was a good control
- Cas9 seems to appear between the 97 and 191 kDa bands, but closer to the 97 kDa band, which is expected (~114 kDa is what we’re going for)
- Each of the “Cas9 Assembled” lanes have a lot more bands than expected, and this could be attributed to truncated protein expression and cleavage sites
- Kelly can give us cells we can run a western on as a control, to see if the extra bands on lanes 5-7 are attributed to noise
Sam
- Interviewed Dr. Postelnick
- Emailed Dr. Tullman-Ercek
- Cited antibiotics pamphlet for adults
Tasfia
- DpnI digest of Cas9-Lrz-SS PCR products from 8.29.16
- Golden Gate PCR for sfGFP and mCherry (nine reactions, one 50-uL reaction for each)
- sfGFP taken from 8.23.16 miniprep
- mCherry taken from 7.35.16 miniprep
- Amount of template used:
- Conditions: 95°C (5:00) | 95°C (0:07), 54°C (0:10), 72°C (0:43) | 72°C (2:00)
- Ran gels on Tet-Lrz-GFP (8.29.16), Cas9-Lrz-SS (8.29.16), and all the GFP/mCherry for GG PCR products (8.30.16)
- Used SybrSafe from Jewett lab (bands were very faint on all gels)
- Recommended: Use ~3 uL SybrSafe on small 10-well gels (with SybrGreen you can get away with ~1 uL)
- These gels used ~3 uL on the 14-well gels
2kB band is very faint
Tyler
- Redesigned primers for restriction digestion of Cas9 into PSB1C3
- Looked into reusing primers for sequencing of gRNA
- Transformation test with Cam plates
- Cam and Tet plate test results
- Tet plates prepared with Kelly’s LB grew exponentially more colonies than tet plates prepared with our LB on August 8th
- gRNA Gibson product grew one colony
- Nothing grew on plates prepared with our LB, plates prepared with Kelly’s LB, or Kelly’s already-prepared plates from Leonard lab
- There’s probably something wrong with our positive control plasmid for Cam
- Drafted e-mail to Tullman-Ercek w/Sam
Jordan
- Started cytosolic GFP cultures for tomorrow
- Interviewed Dr. Postelnick
- Worked on travel grant
- Read instructions for millipore filters
Michelle
- Ran western blot
- We successfully expressed Cas9
- There are no bands in lanes 2-4 because Thush didn’t induce the promoter but Ben offered to run another Western after inducing
- It was a good control
- Cas9 seems to appear between the 97 and 191 kDa bands, but closer to the 97 kDa band, which is expected (~114 kDa is what we’re going for)
- Each of the “Cas9 Assembled” lanes have a lot more bands than expected, and this could be attributed to truncated protein expression and cleavage sites
- Kelly can give us cells we can run a western on as a control, to see if the extra bands on lanes 5-7 are attributed to noise
Sam
- Interviewed Dr. Postelnick
- Emailed Dr. Tullman-Ercek
- Cited antibiotics pamphlet for adults
Tasfia
- DpnI digest of Cas9-Lrz-SS PCR products from 8.29.16
- Golden Gate PCR for sfGFP and mCherry (nine reactions, one 50-uL reaction for each)
- sfGFP taken from 8.23.16 miniprep
- mCherry taken from 7.35.16 miniprep
- Amount of template used:
- Conditions: 95°C (5:00) | 95°C (0:07), 54°C (0:10), 72°C (0:43) | 72°C (2:00)
- Ran gels on Tet-Lrz-GFP (8.29.16), Cas9-Lrz-SS (8.29.16), and all the GFP/mCherry for GG PCR products (8.30.16)
- Used SybrSafe from Jewett lab (bands were very faint on all gels)
- Recommended: Use ~3 uL SybrSafe on small 10-well gels (with SybrGreen you can get away with ~1 uL)
- These gels used ~3 uL on the 14-well gels
2kB band is very faint
Tyler
- Redesigned primers for restriction digestion of Cas9 into PSB1C3
- Looked into reusing primers for sequencing of gRNA
- Transformation test with Cam plates
- Cam and Tet plate test results
- Tet plates prepared with Kelly’s LB grew exponentially more colonies than tet plates prepared with our LB on August 8th
- gRNA Gibson product grew one colony
- Nothing grew on plates prepared with our LB, plates prepared with Kelly’s LB, or Kelly’s already-prepared plates from Leonard lab
- There’s probably something wrong with our positive control plasmid for Cam
- Drafted e-mail to Tullman-Ercek w/Sam
