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