Thursday, September 15th
Tasks:
Michelle
- Streaked JC8031 from glycerol stocks on Amp, Kan, Cam, Tet plates
- Helped Tyler pick Golden Gates for AmiA
- Plated cells for Cas9-gRNA cotransformation
Sam
- Printed Auburn Gresham sheets
- Updated OneNote
- To do: design Auburn Gresham survey
Tasfia
- Website content
- Restriction Digest for Cas9 into pSB1C3 for part submission
- Cas9 restriction digest
- 4 μL Cas9 with added restriction sites (named “Cas9-Res-Dig,” 248.2 ng/μL)
- 1 μL EcoRI-HF
- 1 μL SpeI GQ
- 5 μL 10X Cutsmart buffer
- 39 μL nuclease-free water
- Run for 60 minutes at 37°C, heat inactivated enzymes at 80°C for 20 minutes
- pSB1C3 restriction digest
- 4 μL linearized pSB1C3 (25 ng/μL)
- 4 μL “Master Mix” as instructed by iGEM protocol
- 2.5 μL Cutsmart buffer (looking back, should have used NEB Buffer 2)
- 0.25 μL EcoRI-HF
- 0.25 μL SpeI GQ
- 9 μL nuclease-free water
- 0.25 μL DpnI
- Run for 30 minutes at 37°C, heat inactivated enzymes at 80°C for 20 minutes
- Ligated Cas9 with pSB1C3 (two reactions to see which protocol works, if any)
- iGEM ligation protocol
- 2 μL pSB1C3 after restriction digest
- 4.1 μL Cas9 digest (2:1 insert-to-backbone molar ratio)
- 1 μL T4 ligase buffer
- 0.5 μL T4 ligase
- 2.4 μL nuclease-free water
- Incubated at 16°C for 30 minutes, heat inactivated at 80°C for 5 minutes
- Tyo lab ligation protocol
- 4 μL pSB1C3 after restriction digest
- 8.2 μL Cas9 digest (2:1 insert-to-backbone molar ratio)
- 2 μL T4 ligase buffer
- 1 μL T4 ligase
- 4.8 μL nuclease-free water
- Incubated at room temperature for 20 minutes, no heat inactivation (immediate transformation)
- Transformed ligated products
- 2 μL iGEM protocol ligated product
- 2 μL Tyo protocol ligated product
- 1 μL positive transformation control
Tyler
- Picked colonies from GG plates
- Talked to Jarod/picked up maxipreps that can be used for minipreps
Michelle
- Streaked JC8031 from glycerol stocks on Amp, Kan, Cam, Tet plates
- Helped Tyler pick Golden Gates for AmiA
- Plated cells for Cas9-gRNA cotransformation
Sam
- Printed Auburn Gresham sheets
- Updated OneNote
- To do: design Auburn Gresham survey
Tasfia
- Website content
- Restriction Digest for Cas9 into pSB1C3 for part submission
- Cas9 restriction digest
- 4 μL Cas9 with added restriction sites (named “Cas9-Res-Dig,” 248.2 ng/μL)
- 1 μL EcoRI-HF
- 1 μL SpeI GQ
- 5 μL 10X Cutsmart buffer
- 39 μL nuclease-free water
- Run for 60 minutes at 37°C, heat inactivated enzymes at 80°C for 20 minutes
- pSB1C3 restriction digest
- 4 μL linearized pSB1C3 (25 ng/μL)
- 4 μL “Master Mix” as instructed by iGEM protocol
- 2.5 μL Cutsmart buffer (looking back, should have used NEB Buffer 2)
- 0.25 μL EcoRI-HF
- 0.25 μL SpeI GQ
- 9 μL nuclease-free water
- 0.25 μL DpnI
- Run for 30 minutes at 37°C, heat inactivated enzymes at 80°C for 20 minutes
- Ligated Cas9 with pSB1C3 (two reactions to see which protocol works, if any)
- iGEM ligation protocol
- 2 μL pSB1C3 after restriction digest
- 4.1 μL Cas9 digest (2:1 insert-to-backbone molar ratio)
- 1 μL T4 ligase buffer
- 0.5 μL T4 ligase
- 2.4 μL nuclease-free water
- Incubated at 16°C for 30 minutes, heat inactivated at 80°C for 5 minutes
- Tyo lab ligation protocol
- 4 μL pSB1C3 after restriction digest
- 8.2 μL Cas9 digest (2:1 insert-to-backbone molar ratio)
- 2 μL T4 ligase buffer
- 1 μL T4 ligase
- 4.8 μL nuclease-free water
- Incubated at room temperature for 20 minutes, no heat inactivation (immediate transformation)
- Transformed ligated products
- 2 μL iGEM protocol ligated product
- 2 μL Tyo protocol ligated product
- 1 μL positive transformation control
Tyler
- Picked colonies from GG plates
- Talked to Jarod/picked up maxipreps that can be used for minipreps
