Team:Northwestern/08 05

Notebook

Friday, August 5^th

Tasks:

Michelle

Made 50 μL aliquots of nfH₂O
Made more femtomole dilutions
- Tet backbone: 65.1 ng/μL stock = 50.17 fmol
  - 7.97 μL of stock to 10 μL
- mCherry 1: 65.5 ng/μL stock = 132.5 fmol
  - 3.02 μL stock to 10 μL
- TorA: 100μM stock
  - First 1:100 dilution (done to 50 μL)
  - Second 1:25 dilution (done to 50 μL)
Transformed reactions
- Transformed 5 μL of DNA solution to 50 μL of cells for all transformations
- Recovered in 200 μL of SOC media
- Transformation controls:
  - NEB cells: mRFP in pSB1C3 (Cam plate)
  - Our cells:
    - mRFP in pSB1C3 (Cam plate)
    - mRFP in pSB1T3 (not plated because we ran out of Tet plates)
- Golden Gate reactions:
  - NEB cells: mCherry 1 + TorA (Cam plate)
  - Our cells:
    - mCherry 1 + TorA (Cam plate)
    - mCherry 1 + TorA without ligase (Cam plate)
    - Just linearized Tet backbone, no insert (Cam plate)
- Gibson reactions:
  - NEB cells: Gibson for Cas9 (Cam plate)
  - Our cells:
    - Gibson for Cas9 (Cam plate)
    - Gibson kit positive control (Amp plate)
- Golden Gate storage vector ligation reaction:
  - NEB cells: mCherry 1 (Tet plate)
  - Our cells:
  - mCherry 1 (not plated because we ran out of Tet plates)
  - mCherry 2 (Tet plate)
  - mCherry 3 (Tet plate)
  - mCherry 4 (Tet plate)
  - mCherry 5 (Tet plate)
  - GFP 1 (Tet plate)
  - GFP 2 (Tet plate)
  - GFP 3 (Tet plate)
  - GFP 4 (Tet plate)

Sara

Made cam plates
- 300 mL LB
- 4.5 g bacto agar
- Autoclave, then 300 uL Cam

Shu

Gibson assembly with Tyler
- Cas9 Part
  - 5.8µL of water
  - 2.4µL backbone
  - 0.93 µL Cas9-1
  - 0.86µL Cas9-2
  - 10µL of master mix
- Positive Control
  - 10µL of positive control
  - 10µL of master mix

Tasfia

Ran restriction digest of Gibson Assembly Cas9 products with XbaI (25-μL reactions)

“Cas9” (conc’n: 1370 ng/μL; 260/280: 2.33; 260/230: 1.79)
- 0.75 μL DNA
- 2.5 μL Cutsmart 10x buffer (#B7204S exp 6/17)
- 1 μL XbaI
- 20.75 μL nf water
1:3 backbone-to-insert product (conc’n: 1102 ng/μL; 260/280: 2.34; 260/230: 1.83)
- 0.93 μL DNA
- 2.5 μL Cutsmart 10x buffer
- 1 μL XbaI
- 20.57 μL nf water
1:2 backbone-to-insert product (conc’n: 1080 ng/μL; 260/280: 2.33; 260/230: 1.78)
- 0.95 μL DNA
- 2.5 μL Cutsmart 10x buffer
- 1 μL XbaI
- 20.55 μL nf water
- Incubated digest for ~45 minutes at 37°C

Results:

Gibson Product	Concentration (ng/uL)	260/280	260/230
Cas9	50.2	2.10	0.77
1:3	47.8	1.95	0.54
1:2	47.5	1.91	0.51

Ran a gel on digest products, Gibson inserts, and some golden gate products (Thush)
- (mCherry1 (tat?), mCherry2 (tat? first row on the GG purple tube holder), mCherry1 control
- 6X blue loading dye (NEB #B7021S, exp 6/14) was used
  - Wells 1 and 10: Two ladders ~ 1:3 2-log purple ladder-to-loading dye dilution (8 μL per well)
  - Well 2: 1:2 backbone-to-insert Gibson product (6 μL)
  - Well 3: 1:3 backbone-to-insert Gibson product (6 μL)
  - Well 4: “Cas9” Gibson product (6 μL)
  - Well 5: Cas9(1) insert (9.6 μL)
  - Well 6: Cas9(2) insert (10.8 μL)
  - Well 7: mCherry1 tat (6 μL)
  - Well 8: mCherry2 tat (6 μL)
  - Well 9: mCherry1 control (6 μL)
- Ran at 95 V for 48 minutes
Gel looked strange; Gibson products appeared nowhere on the gel, even though the NanoDrop picked up good concentrations.

Troubleshooting
- We let the gel harden too long
- Our ladder is just bad
- Cas9(1) and Cas9(2) show up at ~3kb, when they should be at 1.8 and 1.6, respectively
- Gibson product has a lot of contaminants (low 260/230 ratios on all three samples)
- Diagonal line for “lighter” mCherry bands due to unequal distribution of electric field
- GG products were also not linearized
- Didn’t NanoDrop that before loading gel either

Tyler

Gibson assembly with Shu
Helped with Michelle's transformations