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