Monday, July 25th
Agenda:
- Check the GG storage vector
- Freezer inventory
- Figure out sequencing of Cas9
- Review constructs
- Human practices
- Find protocols for next steps
- Design sequencing primers for the GFP construct
- Ligate GFP and mCherry parts into the storage vector
- Look into Weinberg and Provost travel grants
Tasks:
Jordan
- Discovered pSB1A3 contains BsaI site, not suitable for GFP storage vector
- Looked for periplasmic lysis protocols
- Scheduled call with Dr. Persell
Michelle
- Freezer inventory & organization
Paul
- Figured out sequencing submission process
- Planned ligation
- Planned gRNA plasmid
Sam
- Emailed profs regarding Eligo Bioscience
- Prepped questions for Dr. Persell
- Website work
- Troubleshot gel imager
- Proposed that gels should be ran at a lower voltage for longer duration in order to prevent “band smiling”
Sara
- PCR of GFP and mCherry
- Miniprep of overnight BB part cultures:
- GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44
- mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01
- Procedure:
- 2.5 uL 10X PCR buffer
- 0.5 uL 10 mM DNTPs
- 0.5 uL of forward primer at 10 mM
- 0.5 uL of reverse primer at 10 mM
- 0.5 uL template (Shu's miniprep GFP and mCherry products)
- 0.25 Taq polymerase
- 20.25 uL H20
- PCR settings:
- Denature: 95 C, 7 s.
- Anneal: 51 C, 43 s.
- Elongate 72 C, 2 m.
- Made 400 mL 1% agarose with Shu
- Poured 2 gels
- Ran a gel of the PCR products
- GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest
- We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE
Shu
- PCR of GFP and mCherry
- Miniprep of overnight BB part cultures:
- GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44
- mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01
- Procedure:
- 2.5 uL 10X PCR buffer
- 0.5 uL 10 mM DNTPs
- 0.5 uL of forward primer at 10 mM
- 0.5 uL of reverse primer at 10 mM
- 0.5 uL template (Shu's miniprep GFP and mCherry products)
- 0.25 Taq polymerase
- 20.25 uL H20
- PCR settings:
- Denature: 95 C, 7 s.
- Anneal: 51 C, 43 s.
- Elongate 72 C, 2 m.
- Made 400 mL 1% agarose with Sara
- Poured 2 gels
- Ran a gel of the PCR products
- GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest
- We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE
Tasfia
- PCR on miniprepped INP
- Ran gel on INP PCR product
- Web content
Tyler
- Designed gRNA template w/homology to mRFP plasmid, mRFP insert with correct GG sticky ends, reverse primer
- Sponsors comm (Bio-rad)
Jordan
- Discovered pSB1A3 contains BsaI site, not suitable for GFP storage vector
- Looked for periplasmic lysis protocols
- Scheduled call with Dr. Persell
Michelle
- Freezer inventory & organization
Paul
- Figured out sequencing submission process
- Planned ligation
- Planned gRNA plasmid
Sam
- Emailed profs regarding Eligo Bioscience
- Prepped questions for Dr. Persell
- Website work
- Troubleshot gel imager
- Proposed that gels should be ran at a lower voltage for longer duration in order to prevent “band smiling”
Sara
- PCR of GFP and mCherry
- Miniprep of overnight BB part cultures:
- GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44
- mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01
- Procedure:
- 2.5 uL 10X PCR buffer
- 0.5 uL 10 mM DNTPs
- 0.5 uL of forward primer at 10 mM
- 0.5 uL of reverse primer at 10 mM
- 0.5 uL template (Shu's miniprep GFP and mCherry products)
- 0.25 Taq polymerase
- 20.25 uL H20
- PCR settings:
- Denature: 95 C, 7 s.
- Anneal: 51 C, 43 s.
- Elongate 72 C, 2 m.
- Miniprep of overnight BB part cultures:
- Made 400 mL 1% agarose with Shu
- Poured 2 gels
- Ran a gel of the PCR products
- GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest
- We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE
Shu
- PCR of GFP and mCherry
- Miniprep of overnight BB part cultures:
- GFP concentration: 39.7 ng/uL, 260/280: 2.02, 260/230: 1.44
- mCherry concentration: 38 ng/uL, 260/280: 1.9, 260/230: 1.01
- Procedure:
- 2.5 uL 10X PCR buffer
- 0.5 uL 10 mM DNTPs
- 0.5 uL of forward primer at 10 mM
- 0.5 uL of reverse primer at 10 mM
- 0.5 uL template (Shu's miniprep GFP and mCherry products)
- 0.25 Taq polymerase
- 20.25 uL H20
- PCR settings:
- Denature: 95 C, 7 s.
- Anneal: 51 C, 43 s.
- Elongate 72 C, 2 m.
- Miniprep of overnight BB part cultures:
- Made 400 mL 1% agarose with Sara
- Poured 2 gels
- Ran a gel of the PCR products
- GFP3 and 4 did not have bands in the correct place, so we reran PCR on them and on GFP 2, which had a lower concentration and 260/230 ratio than the rest
- We plan to troubleshoot by changing the voltage we’re running at and using newly diluted TAE
Tasfia
- PCR on miniprepped INP
- Ran gel on INP PCR product
- Web content
Tyler
- Designed gRNA template w/homology to mRFP plasmid, mRFP insert with correct GG sticky ends, reverse primer
- Sponsors comm (Bio-rad)
