Monday, August 29th
Results:
While GFP and the Gibson kit positive control had colonies, the gRNA Gibson product (Tet) and positive transformation control (Cam) did not yield colonies.
The gRNA Gibson has falied grow the past two times we transformed it, so we’re suspicious that it might not be a problem with the Gibson assembly, because the Gibson control has grown on Amp.
Figure 1: GFP transformation from iGEM kit
Figure 2: Gibson positive control on Amp
Tasks:
Jordan
- Made Cam and Tet plates from the LB Kelly gave us
- Two bottles of 250 mL each
- Added 3.75 grams of Bacto agar to each bottle and autoclaved, cooled to 55°C in water bath
- Added 250 uL of Cam and Tet to respective media
- Poured approximately 20 plates of each
- Wrapped in foil and placed in the fridge
Michelle
- Troubleshot transformation plating
- Started running a western blot on TetR-Cas9 and Assembled Cas9 (in triplicate) with Ben from the Jewett lab
Sam
- Worked on questions for Dr. Postelnick
- To do: set up a meeting (or maybe just email?) Dr. Tullman-Ercek
- Ask whether she knows of any good delivery mechanisms to get large proteins to the periplasm
- Cas9 size
- What pathways we’re already trying
Tasfia
- Went to Jewett lab for western blotting TetR-Cas9 and Assembled Cas9
- PCR: Linearization of tet backbone for GFP
- Three 50-uL reactions
- 1 uL OneTaq 2X Master Mix
- 1 uL DMSO
- 1 uL 10 uM forward primer
- 1 uL 10 uM reverse primer
- 1 uL template
- Template concentration: 28 ng/uL
- Template amounts used:
- 28 ng
- 2.8 ng
- 1.4 ng
- Conditions:
- Products are DpnI digesting at room temperature overnight
- PCR: Linearization of Cas9 for signal sequences
- We had to rerun the Cas9-Lrz-SS procedure because the gel extractions from the most recent PCR gave us erroneous concentrations and 260 ratios (~555 ng/uL, really high 260/280 and 260/230 ratios)
- 25 uL OneTaq 2X Master Mix
- 1 uL DMSO
- 1 uL 10 μM forward primer
- 1 uL 10 uM reverse primer
- 1 uL template
- 21 uL nuclease-free water
- Two Cas9 template samples used:
- “Cas9 1:3” (concentration ~155 ng/uL)
- “Cas9 miniprep w/ antibiotic” (concentration ~30 ng/uL)
- Four 50-uL reactions with the following template amounts
- “Cas9 1:3”
- 15 ng
- 1.5 ng
- “Cas9 miniprep w/ antibiotic”
- 3.0 ng
- 1.5 ng
- Conditions:
- Product is in the thermal cycler and will be ready to DpnI digest tomorrow morning
Tyler
- PCR: Linearization of mRFP
- Aided in western blotting
- Ordered Gel Kit & SybrSafe
- Troubleshot nanodrop
- Took some plate pics
