Notebook

Friday, July 29^th

Tasks:

Jordan

Gel purified the linearized tet backbone
- Mass of gel extract ~300 mg, used 900 uL of GEX buffer
- Eluted with water, 30 uL, stood for 5 minutes
- Nanodrop readings: 13.6 ng/ul, 260/280: 1.93, 260/230: 0.35
Submitted payment info to Experiment so we should be sent a check soon
Transformed Gibson product
- Thawed cells for ~15 mins
- 5µL of each Gibson product into 50 µL of competent cells
- On ice for 30 mins
- Heat shocked for exactly 45 seconds
- Ice for 5 mins
- 450 µL of SOC into each

Michelle

Ran gel of Tet linearized backbone
- 25 uL PCR rxn + 5 uL 6X Blue Gel Loading Dye
- 2 uL ladder + 6 uL 6X Blue Gel Loading Dye
Reran PCR Linearization of the Tet Backbone for Cas9 Gibson/test of Taq and master mix
- OneTaq Master Mix + 0.5 uL primers
  - 10.5 uL water
  - 1.0 uL Tet template (from miniprep)
  - 0.5 uL 10uM fwd primer
  - 0.5 uL 10uM rev primer
  - 12.5 uL OneTaq Master Mix
- Separate PCR mix components + 0.5 uL primers
  - 19.75 uL water
  - 2.5 uL 10X standard buffer
  - 1.0 Tet template (from miniprep)
  - 0.5 uL 10mM dNTPs
  - 0.5 uL 10uM fwd primer
  - 0.5 uL 10uM rev primer
  - 0.25 uL Taq polymerase
- OneTaq Master Mix + 1.25 uL primers
  - 9.0 uL water
  - 1.0 uL Tet template (from miniprep)
  - 1.25 uL 10uM fwd primer
  - 1.25 uL 10uM rev primer
  - 12.5 uL OneTaq Master Mix
- Separate PCR mix components + 1.25 uL primers
  - 18.25 uL water
  - 2.5 uL 10X standard buffer
  - 1.0 Tet template (from miniprep)
  - 0.5 uL 10mM dNTPs
  - 1.25 uL 10uM fwd primer
  - 1.25 uL 10uM rev primer
  - 0.25 uL Taq polymerase
- Control without Template
  - 11.5 uL water
  - 12.5 uL OneTaq Master Mix
  - 0.5 uL 10uM fwd primer
  - 0.5 uL 10uM rev primer
- 95°C (2:00) | 95°C (0:07), 60°C (0:10), 72°C (1:06) [10 cycles] | 95°C (0:07), 50°C (0:10), 72°C (1:06) [18 cycles] | 72°C (5:00)
- 25 μL reaction volume, 28 cycles
Ran gel on the Tet RTW-PCR troubleshooting iteration

Gel Extraction of the Linear Tet Backbone
- Gel Extracted 1 (lane 4) and 3 (lane 8)
- #1: weight of gel excised: 106.9 mg
- #3: weight of gel excised: 163.4 mg
- Used Epoch kit & team-modified procedure

Sara

Ran a colony PCR on the GFP and mCherry/storage vectors with Shu
- Used Vf and Vr primers, 25 uL total volume, 12.5 uL OneTaq MM. Ran 3 controls: No DNA- the PCR reaction contained everything but template DNA. Positive control- DNA straight from the iGEM kit containing RFP, which is a similar size to our desired inserts (J04450 in pSB1K3), and Backbone- Which was only the pSB1K3 backbone
- 12.5 uL OneTaq MM
- 0.5 uL BB Vf Primer
- 0.5 uL BB Vr Primer
Ran a gel on the colony PCR
- The results look like the products are all too small to say that the inserts were successfully placed into the storage vector, but we didn’t run any controls, so we can’t be absolutely positively sure

Tyler