Team:Northwestern/08 04

Notebook

Thursday, August 4th

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

  • Made cam plates
    • 275 mL LB
    • 4.5 g bacto agar
    • Autoclave, then 275 uL Cam
  • Emailed Quentin about getting 6X purple loading dye from the Jewett lab

Shu

  • Retransform the Gibson products in the freezer
    • 3uL 1:2 product (Shu made)
    • 3uL 1:3 product (Shu made)
    • 3uL product (Jordan made)
    • 1uL positive control from competent cell kit (in J04450- pSB1C3)
    • 1uL positive control from Gibson kit
    • 1uL water
    • 200µL of SOC (as per Kelly’s protocol)
    • Plated the entire tube

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