Notebook

Thursday, August 4^th

Tasks:

Jordan

Transformed the Golden Gate projects:
- Left on ice for ten seconds, then all reactions were pipetted into cells within one minute
- Added 600 uL SOC (realized I left some tubes off ice for a sec while I was adding media but that hopefully shouldn’t be a problem)
- Plated all 650 uL
Transformed GFP/MCherry storage vectors:
- Used 600 uL of SOC compared to usual 450

Michelle

GFP Gel extraction
Made more femtomole dilutions
- Tet backbone: made 25 μL more
  - 11.84 μL of 27.4 ng/μL stock to 12.5 μL
  - 4.35 μL of 37.3 ng/μL stock to 6.25 μL
  - 4.41 μL of 36.8 ng/μL stock to 6.25 μL
- GFP1: made 10 μL more
  - 2.74 μL of 72.2 ng/μL stock to 10 μL
- GFP2: made 10 μL more
  - 2.30 μL of 86.0 ng/μL stock to 10 μL
PCR Tet backbone to linearize for GFP in case this golden gate doesn’t work
- 2 x 50 μL reactions
  - 20 μL nfH₂O
  - 1 μL DMSO
  - 2 μL Tet backbone template (from miniprep)
  - 1 μL 10μM forward primer
  - 1 μL 10μM reverse primer
  - 25 μL OneTaq Master Mix
- 1 negative control (no template)
  - 22 μL nfH₂O
  - 1 μL DMSO
  - 1 μL 10μM forward primer
  - 1 μL 10μM reverse primer
  - 25 μL OneTaq Master Mix
Poured a gel to run the Tet linearization for GFP
- ~20–30 mL of 1% agarose
- 2 μL SybrGreen
DpnI digested the Tet linearized for GFP PCR product
- 0.5 μL of DpnI added to each 50 μL PCR tube
- Did not digest the negative control tube
Plated ligation products of the Golden Gate parts into storage vectors

Sara

Shu