Tuesday 11/05/13

Posted on November 5, 2013 by admin

10:00a – 12:30a

Probe Making

New short probes

  1. D07 9 kb Green transcribed
  2. E02 13 kb green (between R/Y)
  3. E03 BLUE 13 KB (no genes, good PC/Psc)
  4. E04 BLUE 10 Kb (flanked by strong PolII)
  5. E05 BLACK/blue 54 kb region upstream of E(Pc)
  6. F08 RED 7 kb region next to black 1, tRNA locus
  7. F12 Taf1_(17kb yellow/green)
  8. G07 AlphaTub84 (5 kb yellow)

Master Mix

  • 6 pmol/uL common primer (6 uM). want 25 pmol = 4 uL per reaction. 4*9 = 36 uL
  • 25*9 = 225 uL Phusion. + 5 uL 1:20 library + 180 uL ddH2O.
  • nanodrop all primers (fresh 1:10 dilutions). All close to 300 ng/uL. Forgot to use single-stranded read measurement!

Test gel

  • samples 1-8 on the gel and 1-8 in the nanodrop plots are the new short probes from above. Other nanodrop results are from yesterday.
    newShortProbes

shortProbe2PCR

shortProbePlots

moreProbesPlots

moreProbesPCR

T7 reactions

  • setup new T7 reactions with 1.5 uL buffer per reaction (instead of 2 uL). 8 new short reactions + 6 from yesterday.
  • running O/N.

Meetings

  • team meeting before meeting with XZ to discuss Hao’s project

STORM

  • E10 RNA. Did not score cells, will have to do multiple rounds of imaging
  • qualitative observation: substantial variation in dot size (STORM) and intensity of RNA, greater than DNA region.
  • issues with focus on E10 RNA / STORM4. Still getting regions of field of view that are black holes.
  • Made 3 new 500 uL resealable chambered slides.
  • attempting to fix quadview on STORM2 — apetures substantially out of alignment.

STORM analysis

For tomorrow

  • Talk to Hao about EDAC carboxyl bead crosslinking
  • Talk to Hao about HPLC cy7
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