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
- mock
- PSC
- PPES (Pc, Psc, Esc, Scm)
- Ph-P + Ph-D
- Pho
- Su(Z)2
- 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