Michelle

• Gel extracted gRNA and signal sequences
• Used IBI protocol (kit from Leonard Lab)
• Nanodrop results: all under 10 ng/uL, 260/280 above 1.9, 260/230 around 0.5 — discarded
• Reran signal sequence PCR (to add homology)
• 1 uL template
• 1 uL fwd primer 10uM
• 1 uL rev primer 10uM
• 1 uL DMSO
• 21 uL nfH₂O
• 25 uL OneTaq Master Mix
• Ran 2 gels on the signal sequence PCR products
  
  

  

Tasfia

• DpnI digested Cas9-Lrz-SS PCR product
• Made more 10X TAE
• Registered all the ChemEs and Jordan for iGEM Jamboree
• DpnI digested Tet-Lrz-GFP for two hours and ran a gel screen on it (with 1 uL of our remaining SybrGreen)
  
• Only primer dimers result from the Tet-Lrz-GFP PCR

Tyler

• Gibson Assembly of gRNA-mRFP
• gRNA-mRFP Assembly
• 0.31 µL insert
• 0.4 µL backbone
• 4.29 µL water
• 5 µL Gibson mix
• Negative Control
• 0.4 µL backbone
• 4.6 µL water
• 5 µL Gibson Mix
• Quantities transformed (heat shocked 30s, 200µL SOC)
• 3 µL gRNA-mRFP assembly product
• 2 µL negative control
• 2µL positive Gibson kit control
• 1µL positive transformation control