Tuesday 07/14/15

10:00 am – 8:00 pm

Goals

  • make cDNA from RNAi samples from last week
  • qPCR of RNAi samples from last week
  • set up more RNAi for next week
  • make more dsRNA for RNAi
  • reply to Hao, comments on essay
  • analyze Lib15 + 16 data
  • chromatic corrections for L15 data from last week.

Cell culture

  • heat-inactivate fresh aliquot of FBS: 30 min at 56 C with mixing (timing important)
  • DGRC says can’t use Sneider’s from Sigma (http://www.flyrnai.org/DRSC-PRC.html)
  • Made up new BG3 media: 10 ug/mL insulin + 10% heat-inactivated FBS + Schneider’s (from Life Sciences)
  • set up new RNAi. Out of Psc (and not sure it was working anyway)
  • passaged BG3-cells. Set up large flask to grow enough cells to freeze stocks next week.

First strand cDNA synthesis

RNA dilutions (master 1)

  • (desired 10 ug of RNA) -> 20 uL RNA (have now ~200 ng/uL instead of 2500 ng/uL)
  • 2 uL oligo-dT primer (20 uL)
  • 2 uL dNTP mix (20 uL)
  • heat to 65C for 5 min, return to ice.

per reaction (master 2)

  • 4 uL 10x buffer (40 uL)
  • 8 uL MgCl2 (80 uL)
  • 4 uL DTT (40 uL)
  • 2 uL RNase OUT (20 uL)
  • 2 superscript3 (20 uL)

sample order

  1. mock
  2. PSC
  3. PPES (Pc, Psc, Esc, Scm)
  4. Ph-P + Ph-D
  5. Pho
  6. Su(Z)2
  7. HS (heat shock)
    8 RT (room temp cntrl)

QRTPCR reactions

  • Per well, 2.5 uL of chosen primer mix, 2.5 uL of cDNA
  • Per well master
    • 12.5 uL Hot Start Phusion
    • 1.25 uL Eva Green
    • 6.25 uL water
  • 100x total master
    • 1250 Phusion
    • 125 Eva Green
    • 625 uL water
    • 20 uL each
  • running @ 7:33 PM

MERFISH

Chromatic corrections

  • looks like alignment is already pretty good and that accuracy is limited by spot-fitting (of non-point source RNA foci)
  • see Evernote notebook
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