Tuesday, September 6th
Tasks:
Jordan
Figure 1: Troubleshooting summary
- Ran periplasm fraction protocol on Cas9 culture in TetR
- Centrifuged 25 mL suspension for 20 minutes at 4000 rpm and 4C
- Discarded supernatant
- Resuspended in 6.25 mL of ice-cold sucrose cell fractioning buffer 1
- Incubated for 20 minutes, shook periodically
- Centrifuged for 30 minutes at 4000 rpm and 4 degrees
- Discarded supernatant
- Resuspended in 1.56 mL ice cold buffer 2 with no detergents, transferred to eppendorf tube
- Incubated for 20 minutes on ice
- Centrifuged for 15 minutes at 11000 rpm on tabletop centrifuge
- Saved supernatant, in fridge
Michelle
- Gel extracted Cas9 for SS and Cas9 for iGEM
- Nanodrop results:
- Cas9 lnrz for SS: 9.0 ng/uL, 260/280: 1.8, 260/230: 0.44
- Cas9 res.dig. for iGEM: 8.8 ng/uL, 260/280: 1.85, 260/230: 0.43
Paul
- Got sequencing back for gRNA assembled construct
- Results aligned under “Assembled Parts>pSB1T3…+gRNA”
- Few point mutations on mRFP and Terminator-likely not too bad
- Gibson assembly of YcdO (10 uL: *6:1 insert:bb, 1% DMSO, 4 hrs @50 C*)
- 4.4 uL of Cas9 lnrz for SS (from 9/1/16 PCR, gel extracted 9/6/16- 9 ng/uL)
- 0.5 uL of YcdO (from PCR products, 38 ng/uL)
- 0.1 uL DMSO
- 5 uL Gibson Assembly HiFi MasterMix
- Met w/ Joe (w/ Tyler) about modeling
- Detect potential off target cleavage sites of our gRNA in the gut microbiome
- Restriction digest linear pSB1C3 and linear Cas9 (EcoRI, SpeI)
Sam
- Sent Emma Nechamkin emails
- Grew up two cultures (25 mL to start, ~22 mL now exist)
- Both JC8031
- One has pClyA-GFP-His6 Cam
- Induced with 290 uL arabinose at about .5 absorbance (actual absorbance labelled)
- One is normal
- No antibiotic
- Overnight in the 37°C shaking incubator
- Found and watched antibiotics and CRISPR videos
- Started adding graphics to pamphlet
Tyler
- Mathematical modeling meeting w/Joe
- Transformation of ligated products + Gibson SS (Ycdo)
- 3µL Ligated Cas9 Product
- 5µL Gibson SS Product
- 1µL Transformation control (50pg mRFP control)
- 2µL Gibson (+) control
Jordan
- Ran periplasm fraction protocol on Cas9 culture in TetR
- Centrifuged 25 mL suspension for 20 minutes at 4000 rpm and 4C
- Discarded supernatant
- Resuspended in 6.25 mL of ice-cold sucrose cell fractioning buffer 1
- Incubated for 20 minutes, shook periodically
- Centrifuged for 30 minutes at 4000 rpm and 4 degrees
- Discarded supernatant
- Resuspended in 1.56 mL ice cold buffer 2 with no detergents, transferred to eppendorf tube
- Incubated for 20 minutes on ice
- Centrifuged for 15 minutes at 11000 rpm on tabletop centrifuge
- Saved supernatant, in fridge
Figure 1: Troubleshooting summary
Michelle
- Gel extracted Cas9 for SS and Cas9 for iGEM
- Nanodrop results:
- Cas9 lnrz for SS: 9.0 ng/uL, 260/280: 1.8, 260/230: 0.44
- Cas9 res.dig. for iGEM: 8.8 ng/uL, 260/280: 1.85, 260/230: 0.43
Paul
- Got sequencing back for gRNA assembled construct
- Results aligned under “Assembled Parts>pSB1T3…+gRNA”
- Few point mutations on mRFP and Terminator-likely not too bad
- Gibson assembly of YcdO (10 uL: *6:1 insert:bb, 1% DMSO, 4 hrs @50 C*)
- 4.4 uL of Cas9 lnrz for SS (from 9/1/16 PCR, gel extracted 9/6/16- 9 ng/uL)
- 0.5 uL of YcdO (from PCR products, 38 ng/uL)
- 0.1 uL DMSO
- 5 uL Gibson Assembly HiFi MasterMix
- Met w/ Joe (w/ Tyler) about modeling
- Detect potential off target cleavage sites of our gRNA in the gut microbiome
- Restriction digest linear pSB1C3 and linear Cas9 (EcoRI, SpeI)
Sam
- Sent Emma Nechamkin emails
- Grew up two cultures (25 mL to start, ~22 mL now exist)
- Both JC8031
- One has pClyA-GFP-His6 Cam
- Induced with 290 uL arabinose at about .5 absorbance (actual absorbance labelled)
- One is normal
- No antibiotic
- Overnight in the 37°C shaking incubator
- Found and watched antibiotics and CRISPR videos
- Started adding graphics to pamphlet
Tyler
- Mathematical modeling meeting w/Joe
- Transformation of ligated products + Gibson SS (Ycdo)
- 3µL Ligated Cas9 Product
- 5µL Gibson SS Product
- 1µL Transformation control (50pg mRFP control)
- 2µL Gibson (+) control
