Jordan

• Cleaned out the fridge, got rid of Interlab stuff, the base Cas9 plates that we have minipreps and glycerol stocks of, and controls and stuff that never worked anyway
• Transformed 3 uL of DsbA Gibson from Gene blocks and from added homology PCRs, negative control with only backbone, and 1ul (50 pg) of competent cell test RFP
• 30 second heat shock, 950 uL SOC, 1 hr 15 minute incubation, plated 2 plates of each

Paul

• Added Golden Gate transformation pictures to slideshow
• Presented at meeting with Profs

Sam

• Emailed Dr. Tullman-Ercek
• Made a gel
• Updated OneNote
• Transformation with Jordan

Sara

• PCR to extend SS homology
• 50 uL reaction of every SS we have
• 0.5 uL template (SS)
• 1.0 uL fwd primer @ 10 mM
• 1.0 uL rev primer @ 10 mM
• 1.0 uL DMSO
• 21.5 uL dH20
• 25 uL Onetaq 2X MM
• PCR purified signal sequences

Tasfia

• PCR: Linearization of Cas9 for signal sequences (again)
• 50 uL reaction of every SS we have
• 1 uL template (“Cas9 1:3 136 ng/uL” nanodropped 133 ng/uL)
• 13 ng (“Cas9-Lrz-SS ‘A’”)
• 1.3 ng (“Cas9-Lrz-SS ‘B’”)
• 1 uL fwd 10 uM primer
• 1 uL rev 10 uM primer
• 1 uL DMSO
• 21 uL nuclease-free water
• 25 uL OneTaq 2X Master Mix with Standard Buffer

• DpnI digested (0.5 μL DpnI per tube) Cas9-Lrz-SS for one hour at 37oC (heat block)
• Nanodrop:
• Before purification:
• Cas9-Lrz-SS “A”: 449.4 ng/uL, 260/280: 1.51, 260/230: 0.66
• Cas9-Lrz-SS “B”: 467.9 ng/uL, 260/280: 1.54, 260/230: 0.69
• After purification:
• Cas9-Lrz-SS “A”: 148.3 ng/uL, 260/280: 1.80, 260/230: 1.32
• Cas9-Lrz-SS “B”: 56.0 ng/uL, 260/280: 1.84, 260/230: 1.29
• Cas9-Lrz-SS product B was used in today’s Gibson reactions
• Made Gibson reaction mixes with DsbA and Cas9-Lrz-SS (5:1 insert-to-backbone molar ratio)
• Gibson assembly #1: DsbA insert with PCR-added homology
• 0.89 uL Cas9-Lrz-SS PCR product from today
• 0.23 uL DsbA insert with added homology PCR’ed today
• 3.88 uL nuclease-free water
• 5.00 uL Gibson master mix
• Gibson assembly #2: DsbA insert gBlock
• 0.89 uL Cas9-Lrz-SS
• 1.67 uL DsbA gBlock
• 2.54 uL nuclease-free water
• 5.00 uL Gibson master mix
• Negative control
• 0.89 uL Cas9-Lrz-SS
• 4.11 uL nuclease-free water
• 5.00 uL Gibson master mix
• Gibson assembly reactions were run at 50°C for one hour and transformed by Jordan and Sam

Tyler

• Gel Screen of SS + Cas9

• Aided with various steps of the cloning procedure (set up thermocyclers, did gibson calculations, etc.)