Monday 12/16/13

9:50a – 1:00a

Group meeting

Probe making

  • cy7 dye from Lumipore arrived
  • A750 labeled IDT primers arrived
  • first test gel — try old gel of Bogdan’s (11/18). Ran smeary and fast, primers ran off the bottom
  • reran with precast gel (forgot to take tape off at start, removed after first hour, so gel ran a bit squished. Not a good day for gels)
  • substantial primer bands still visible, except in lane 5. Why are 2 and 3 the strongest? 1 I guess is a bit spread out. Seem dilution helps.
  • G5 is still reproducibly creating a double band. I don’t like this.
    RTforMultiPrimerRatioTest_1

RTforMultiPrimerRatioTest_2

More RT tests

  • samples 3 + 4
  • 2 uL primer, 6 uL RNA, 3 uL new dNTPs, 65C denature, ice anneal, 42C incubation. Hot Start.
  • 2 uL primer, 6 uL RNA, 3 uL new dNTPs, 65C denature, ice anneal, 50C incubation. Hot Start.
  • (4 uL 5x buffer + 1 uL RNasin ) x5 + 1 uL Maxima

chromatin imaging

STORM results

  • staining failed again! Despite high concentration matches previous successful staining just a few days ago.
  • Maybe the hybe solution got too diluted with 2 probes both added in large volumes?

Cell staining

  • Try 3 uL hybes, with positive controls with water added instead of P3 / S3.
  • F11-P1 (3uL) + .7 uL S1-A647 + 3.7 uL ddH2O.
  • F11-P1 (3uL) + Fll-P3 (3uL) + .7 uL S1-A647 + .7 uL S3-A750
  • Fll-P3 (3uL) + .7 uL S3-A750
  • G1-P1 (3uL) + .7 uL S1-A647 + 3.7 uL ddH2O.
  • G1-P1 (3uL) + Gl-P3 (3uL) + .7 uL S1-A647 + .7 uL S3-A750
  • Gl-P3 (3uL) + .7 uL S3-A750

STORM analysis

  • Analyzing D07 data (9kb green transcribed)
    • highly variable, pretty messy, some spots have lots of density
    • sometimes round symmetric blur, 3K localizations
  • E02, 13 kb green domain
    • concentrated, punctate, symmetric
    • data only goes up to 0_5, missing last 4 cells.
  • Running E11 bead fitting (somehow didn’t get analyzed) on Tuck
  • Running E03 dot and bead fitting on Tuck (started 11pm)
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