Thursday, August 25th
Tasks:
Jordan
- Ran the cell fractions on the plate reader with Michelle
- 485/520 fluorescence (rows = replicates)
- No longer have linearized Cas9 for SS gel extract from 8.19 and 8.22 - lost the DNA in the ethanol precipitation
- Ethanol precipitation of 8.19 and 8.22 linearized cas9 for SS gel extract
- Added 1/10 the sample volume of 3M sodium acetate (2 uL for each 20 uL sample)
- Added 2.5 volumes of 100% ethanol (added 50 uL of 95% ethanol (didn’t make a difference)) to each sample
- Froze for 1 hr at -80°C
- Centrifuge at max speed (14,000 rpm) for 30 minutes
- Decant or pipette off supernatant
- Air dry pellet for 15 minutes (then 30 in heat block)
- Resuspend in water, vortex and spin down
- Negative DNA was nanodropped
- Lost the DNA
Michelle
- Cleared unnecessary items from freezer
- Made a 1000X dilution of Cam
- 0.51 mL of 100 mg/mL stock diluted to 1.5 mL using 200-proof ethanol
- Made more Cam plates
- 200 mL LB + 3.0097 g Bacto Agar (autoclaved)
- PCR signal sequences to add more homology
- Nanodrop:
- FhuD: 13.6 ng/uL
- AmiA: 10.9 ng/uL
- DsbA: 11.3 ng/uL
- YcdO: 18.6 ng/uL
- TorA: 6.5 ng/uL
- NapA: 10.0 ng/uL
- Mix:
- 0.5 uL of template (SS)
- 1.0 uL of fwd primer 10 uM
- 1.0 uL of rev primer 10 uM
- 1.0 uL of DMSO
- 21.5 uL of nfH2O
- 25 uL of OneTaq 2X Master Mix
- Conditions:
- Cut out gRNA and SS bands and froze them
- 200 mL LB + 3.0097 g Bacto Agar (autoclaved)
- Ran plate reader on periplasm fractions with Jordan
Paul
- Tested cell fractioning procedure from Bielfeld iGEM 2014 with Tasfia
- 2 x 20 mL of DeLisa line
- 2 x 20 mL of ClyA-GFP in DeLisa line
- Tested no SDS and SDS in Cell Frac. Buffer 2
- *Final Supernatant contains periplasmic proteins
- Cas9 Expression Protocol Plan:
- Saturday:
- Inoculate 4x 1 mL cultures for overnight growth (add appropriate antibiotic)
- Inoculate 2 cultures of saCas9 with our glycerol stocks
- Pick 2 colonies from plate in fridge (“TetR, Kelly’s cells) from 8-24-16
- Sunday:
- Inoculate/grow 5 mL cultures up to OD=0.5
- Add 500 uL of grown up cultures to 5 mL LB with antibiotic
- Grow to OD=0.5-0.6 (roughly)
- Put on ice once at correct OD
- Express overnight (~16 hrs) at 37 or 18°CC (1 culture of our cells and one culture of TetR Kelly’s cells at 18, and 1 culture of each cell line at 37)
- Use Mordaq’s incubator for 18°C
- Monday: Take 5 mL cultures to Ben in Jewett lab in morning (~9 am)
Shu
- Ran gel on on Cas9-Lrz-SS:
- Excised gel and froze for two hours, then extracted
- Concentrations: see 8.29.16
Tasfia
- Performed cell fractioning procedure with Paul (see his notes)
- Re-ran PCR for Cas9 linearized for signal sequences (to DpnI digest and gel extract next morning)
- 25 uL OneTaq 2X Master Mix
- 1 uL DMSO
- 1 uL 10 uM forward primer
- 1 uL 10 uM reverse primer
- 1 uL template
- Concentration: 28 ng/uL
- Amounts used:
- 15 ng
- 1.5 ng
- 21 uL nuclease-free water
Tyler
- Ran gRNA and Tet-Lrz-GFP PCRs
Jordan
- Ran the cell fractions on the plate reader with Michelle
- 485/520 fluorescence (rows = replicates)
- No longer have linearized Cas9 for SS gel extract from 8.19 and 8.22 - lost the DNA in the ethanol precipitation
- Ethanol precipitation of 8.19 and 8.22 linearized cas9 for SS gel extract
- Added 1/10 the sample volume of 3M sodium acetate (2 uL for each 20 uL sample)
- Added 2.5 volumes of 100% ethanol (added 50 uL of 95% ethanol (didn’t make a difference)) to each sample
- Froze for 1 hr at -80°C
- Centrifuge at max speed (14,000 rpm) for 30 minutes
- Decant or pipette off supernatant
- Air dry pellet for 15 minutes (then 30 in heat block)
- Resuspend in water, vortex and spin down
- Negative DNA was nanodropped
- Lost the DNA
Michelle
- Cleared unnecessary items from freezer
- Made a 1000X dilution of Cam
- 0.51 mL of 100 mg/mL stock diluted to 1.5 mL using 200-proof ethanol
- Made more Cam plates
- 200 mL LB + 3.0097 g Bacto Agar (autoclaved)
- PCR signal sequences to add more homology
- Nanodrop:
- FhuD: 13.6 ng/uL
- AmiA: 10.9 ng/uL
- DsbA: 11.3 ng/uL
- YcdO: 18.6 ng/uL
- TorA: 6.5 ng/uL
- NapA: 10.0 ng/uL
- Mix:
- 0.5 uL of template (SS)
- 1.0 uL of fwd primer 10 uM
- 1.0 uL of rev primer 10 uM
- 1.0 uL of DMSO
- 21.5 uL of nfH2O
- 25 uL of OneTaq 2X Master Mix
- Nanodrop:
- Conditions:
- Cut out gRNA and SS bands and froze them
- 200 mL LB + 3.0097 g Bacto Agar (autoclaved)
- Ran plate reader on periplasm fractions with Jordan
Paul
- Tested cell fractioning procedure from Bielfeld iGEM 2014 with Tasfia
- 2 x 20 mL of DeLisa line
- 2 x 20 mL of ClyA-GFP in DeLisa line
- Tested no SDS and SDS in Cell Frac. Buffer 2
- *Final Supernatant contains periplasmic proteins
- Cas9 Expression Protocol Plan:
- Saturday:
- Inoculate 4x 1 mL cultures for overnight growth (add appropriate antibiotic)
- Inoculate 2 cultures of saCas9 with our glycerol stocks
- Pick 2 colonies from plate in fridge (“TetR, Kelly’s cells) from 8-24-16
- Sunday:
- Inoculate/grow 5 mL cultures up to OD=0.5
- Add 500 uL of grown up cultures to 5 mL LB with antibiotic
- Grow to OD=0.5-0.6 (roughly)
- Put on ice once at correct OD
- Express overnight (~16 hrs) at 37 or 18°CC (1 culture of our cells and one culture of TetR Kelly’s cells at 18, and 1 culture of each cell line at 37)
- Use Mordaq’s incubator for 18°C
- Monday: Take 5 mL cultures to Ben in Jewett lab in morning (~9 am)
Shu
- Ran gel on on Cas9-Lrz-SS:
- Excised gel and froze for two hours, then extracted
- Concentrations: see 8.29.16
Tasfia
- Performed cell fractioning procedure with Paul (see his notes)
- Re-ran PCR for Cas9 linearized for signal sequences (to DpnI digest and gel extract next morning)
- 25 uL OneTaq 2X Master Mix
- 1 uL DMSO
- 1 uL 10 uM forward primer
- 1 uL 10 uM reverse primer
- 1 uL template
- Concentration: 28 ng/uL
- Amounts used:
- 15 ng
- 1.5 ng
- 21 uL nuclease-free water
Tyler
- Ran gRNA and Tet-Lrz-GFP PCRs
