Thursday 08/13/15

Posted on August 13, 2015 by admin

9:30 am – 7:30 pm, 9:00 pm – 11:45 pm

Chromatin Project RNAi

RT reactions

  • protocol
  • 5 uL gene-specific primer mix
  • 10 uL total RNA, (diluted to 400 ng/uL)
  • 5 uL RT buffer
  • 1 uL dNTPs
  • 1 uL Maxima
  • 2 uL RNasin

master 10x

  • 50 uL RT buffer
  • 10 uL dNTPs
  • 10 uL Maxima
  • 20 uL RNasin

notes

  • noticed precipitate in my RT buffer. opened a new buffer. thawing now.
  • see same thing in new RT buffer. Didn’t notice before, but it’s pretty subtle
  • Primer mix: alpha-Tub84, Act5C, GADPH1, Pc, Ph-p, Antp, Abd-B, en
  • sample order:
    1. Ph 7-7 10 uL, –> 250 ng/uL
    2. mock 7-7 10 uL –> 250 ng/uL
    3. Ph 7-14 2.5 uL –> 210 ng/uL
    4. mock 7-14 2.5 uL –> 210 ng/uL
    5. Ph 8-8 10 uL –> 210 ng/uL
    6. mock 8-8 5 uL –> 450 ng/uL
    7. Pc 8-8 5 uL –> 250 ng/uL
    8. mock 6-29 2.5 uL –> 250 ng/uL

RT clean-up

  • try alkaline hydrolysis followed by oligo column-clean-up.
  • eluted in 20 uL. Spec’d all samples. conc’s above
  • dilute 5 into 20 uL ddH2O. use 2 uL per sample to set up.
  • try running with a no primer control

QPCR

Reaction set-up per well

  • 2 uL diluted DNA ~50 ng/uL
  • 1.25 uL EvaGreen
  • 12.5 uL Phusion 2x master mix
  • 2.5 uL primer combo (fwd/rev)
  • 6.75 uL ddH2O

Master mix (80x)

  • 100 uL EvaGreen
  • 1000 uL Phusion 2x master mix
  • 540 uL ddH2O
  • 20.5 uL per well

Plate setup

  • 8 DNA samples + 1 no template control
  • 8 gene-primer combos for each
  • 72 PCR reactions

Analysis

  • failed!! essentially indistinguishable similar amplification in all wells. We should have clearly orders of magnitude separated samples
  • probably accrued too much PCR product for these reactions in the pipettes etc.

Gel-troubleshooting

  • complete nonsense:
    TroubleshootingNonsense
  • stupid me. I mixed the foward and reverse primers already for the PCR. Can’t use both forward and reverse primers in the RT, this creates a mess.

To do still

  • upload review!
  • analyze SIM data

Other stuff done

  • Psc null cells, finished freezing down stock
  • send back shuttle form for EMBO -DONE
  • checked staining on BXC cell cycle — not that great via turnkey. Should analyze this data further
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