Jordan

• Helped make competent Delisa cells
• Ligated Cas9 and pSB1C3 restriction digests
• 25 ng pSB1C3 (2ul)
• 37 ng Cas9 insert (1.86 uL)
• 1 ul T4 ligase buffer
• 0.5 ul T4 ligase
• 4.64 ul water
• 1:1 insert:vector
• 16°for 30 minutes, heat kill 80°C 20 minutes

Sam

• Electroporated 4 lines of cells with Paul
• Skipped the negative control because the Jewett lab only had 4 electroporation cuvettes

Sara

• Poured a gel to screen the clyA-PET28a PCR
• 3 uL sybersafe
• 5 uL product, 1 uL dye
• 6 uL ladder, 3 uL dye
• The gel showed that the PCR did not work
• Made more 1% agarose (400 mL)

Tasfia

• Made competent cells out of DeLisa strain (with Paul and Jordan)
• Transformed ligation product (Cas9 into pSB1C3 iGEM backbone for part submission), 3 μL DNA
• Transformed pSB1C3 competent cell test plasmid to examine efficiency of DeLisa competent cells
• Picked more colonies of gRNA+Cas9 cotransformation, and with existing overnight cultures made 2.5-mL aliquots to give cultures more air in the tubes

Tyler

• Gibson Reactions
• Cas9
• 0.74 µL backbone (1:2 dilution)
• 1.15 µL insert (1:5 dilution)
• 3.11 µL water
• 5 µL mix
• SS-Cas9 DsbA
• 0.74 µL backbone
• 0.75 µL insert (1:5 dilution)
• 3.51 µL water
• 5 µL mix
• SS-Cas9 Ycdo
• 0.74 µL backbone
• 0.95 µL insert (1:5 dilution)
• 3.31 µL water
• 5 µL mix Backbone
• (-) control
• 0.74 µL backbone
• 4.26 µL water
• 5µL mix