Team:Northwestern/08 30

Notebook

Tuesday, August 30th

Tasks:

Jordan

  • Started cytosolic GFP cultures for tomorrow
  • Interviewed Dr. Postelnick
  • Worked on travel grant
  • Read instructions for millipore filters

Michelle

  • Ran western blot
    • We successfully expressed Cas9
    • There are no bands in lanes 2-4 because Thush didn’t induce the promoter but Ben offered to run another Western after inducing
    • It was a good control
    • Cas9 seems to appear between the 97 and 191 kDa bands, but closer to the 97 kDa band, which is expected (~114 kDa is what we’re going for)
    • Each of the “Cas9 Assembled” lanes have a lot more bands than expected, and this could be attributed to truncated protein expression and cleavage sites
    • Kelly can give us cells we can run a western on as a control, to see if the extra bands on lanes 5-7 are attributed to noise

Sam

  • Interviewed Dr. Postelnick
  • Emailed Dr. Tullman-Ercek
  • Cited antibiotics pamphlet for adults

Tasfia

  • DpnI digest of Cas9-Lrz-SS PCR products from 8.29.16
  • Golden Gate PCR for sfGFP and mCherry (nine reactions, one 50-uL reaction for each)
    • sfGFP taken from 8.23.16 miniprep
    • mCherry taken from 7.35.16 miniprep
    • Amount of template used:
    • Conditions: 95°C (5:00) | 95°C (0:07), 54°C (0:10), 72°C (0:43) | 72°C (2:00)
  • Ran gels on Tet-Lrz-GFP (8.29.16), Cas9-Lrz-SS (8.29.16), and all the GFP/mCherry for GG PCR products (8.30.16)
    • Used SybrSafe from Jewett lab (bands were very faint on all gels)
    • Recommended: Use ~3 uL SybrSafe on small 10-well gels (with SybrGreen you can get away with ~1 uL)
    • These gels used ~3 uL on the 14-well gels

    2kB band is very faint

Tyler

  • Redesigned primers for restriction digestion of Cas9 into PSB1C3
  • Looked into reusing primers for sequencing of gRNA
  • Transformation test with Cam plates
    • Cam and Tet plate test results
      • Tet plates prepared with Kelly’s LB grew exponentially more colonies than tet plates prepared with our LB on August 8th
      • gRNA Gibson product grew one colony
      • Nothing grew on plates prepared with our LB, plates prepared with Kelly’s LB, or Kelly’s already-prepared plates from Leonard lab
      • There’s probably something wrong with our positive control plasmid for Cam
  • Drafted e-mail to Tullman-Ercek w/Sam

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