Tuesday 07/21/15

10:15 am – 8:00 pm

RNAi experiments

Quantifying knockdown efficiencies by qPCR

cDNA

1. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 1
2. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 2
3. Pc v1
4. Pc v2
5. Pc, Esc, Scm,
6. Esc
7. Suz12
8. Scm
9. SCE
10. mock
11. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 0 (from last week)

genes to test

1. Pc
2. Ph-p
3. Esc
4. Scm
5. Suz12
6. SCE
7. Abd-B
8. Antp
9. Ph-d
10. Gapdh1
11. Act5A

Experiment layout

• Note: don’t have primers for Su(Z)12
• see qPCR setup google doc
• 2.5 uL of primer mix
• 2 uL of cDNA
  

Results

• knockdown doesn’t seem to have worked so well this time.
• maybe some issues with the qPCR, will try to run a few lanes again.
  
• Notes
  • col 9 has Psc instead of Scm
  • col 10 has Sce instead of Scm
  • col 12 has ‘Psc, Sce, Ph-D and alpha-tub84a’ instead of ‘Abd-B, Antp, Gapdh1, and Act5c’ respectively

new dsRNA synthesis

• freeze T7 reactions of original PPES combo. Not sure we’re coming back to this.

STORM

• cells very sparse.
• note: the top filter wheel sometimes gets turned. It needs to be in position 1. take off the inspection port. if empty port 3 is visible in the inspection port, the correct port 1 is aligned. The IR focus lock beam bounces off this dichroic. No idea why people swap this out.

New stains

• prepped whole plate of cells
• should make initial density lower still — a lot of cells detach in sheets during the LN2 treatment.
• seems to me the PcG RNAi treated cells detach more than the wt / mock cells do.
• RNase treated just 2 coverslips. Rest still RNA-intact.