Team:Northwestern/08 19

Notebook

Friday, August 19th

Tasks:

Michelle

  • Ran gel on Cas9 linearized for signal sequences
  • Gel extracted the upper crest
    • Pt 1: 0.2561 g (dissolved in 0.85 mL GEX)
    • Pt 2: 0.3051 g (dissolved in 1.00 mL GEX)
    • Nanodrop: 31.4 ng/uL, 260/280: 1.68, 260/230: 1.17
  • PCR for GFP 1 to test template DNA amounts
    • Made a 1:10 dilution of the 42.5 ng/uL miniprepped stock of template
    • Reactions:
      • 1.0 uL of template reaction
        • 1 uL of 4.25 ng/uL GFP template
        • 1 uL of 10 uM forward primer
        • 1 uL of 10 uM reverse primer
        • 1 uL of DMSO
        • 21 uL of nfH2O
        • 25 uL of 2X OneTaq Master Mix
      • 0.5 uL of template reaction
        • 0.5 uL of 4.25 ng/uL GFP template
        • 1 uL of 10 uM forward primer
        • 1 uL of 10 uM reverse primer
        • 1 uL of DMSO
        • 21.5 uL of nfH2O
        • 25 uL of 2X OneTaq Master Mix
      • Conditions: 95°C (5:00) | 95°C (0:07), 54°C (0:10), 72°C (0:43) | 72°C (5:00)
  • Ran gel of GFP test PCR
    • Used NEB Purple Loading Dye 6X
    • 2 uL 2 log purple ladder + 6 uL dye
    • 50 uL PCR reaction + 10 uL dye
    • 15 uL of reaction into each well
    • Results were the same as previous PCRs, did not extract

Paul

  • Did the presentation
  • Looked over Cas9 BB insert primers
  • Looked at GFP primers

Sam

  • Troubleshot GFP PCR. It’s making 500 bp bands instead of 800, so I tried to find other binding sites for the primers. Couldn’t find anything
  • Started work on Resisting Resistance public kit

Tasfia

  • Re-ran PCR/gel for GFP/mCherry for GG, but only for GFP
    • Running with an annealing temperature of 54°C instead of 51°C (which is what we were doing before)
      • NEB recommends an annealing temperature of 53°C, but we ran at 54°C as per Bradley’s suggestion at this morning’s meeting
    • PCR protocol is otherwise exactly the same as the one used the day before (18 August 2016)
  • Ran a gel of GFPs
    • Out of 60 μL PCR product + dye per tube, put 20 μL in each well
    • Did not extract
  • Made progress on “Attributions” web content
  • Made progress on “Project Overview” web content

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