Tuesday 12/10/13

(finally recovering from flu)

11:00a – 9:00p

Probe making

  • run denaturing gel
  • G5 looks weird – what’s with the double band?! maybe give this a test anyway.
  • primers are different lengths, but that doesn’t explain why both primer sets have an additional lower band that doesn’t match the other probes. Major product sizes all look correct.
  • concentrations all in the 1100 ng/uL to 1500 ng/uL range

Cell staining

  • RNase treating cells
  • G5-P1 + G6-P3
  • G1-P1 + G2-P3
  • F12-P1 + G1-P3
  • pre-hybing at 47C

Troubleshooting staining and beads

  • EDAC treatment does seem to reduce staining. Especially long treatment.
  • EDAC treatment (at least on recent samples) does not seem to keep beads attached through the 16 hr hybridization.
  • Maybe EDAC has gone bad? Too much freeze thaw, too long hydrated? Ordered fresh EDAC from invitrogen early last week (12/4, not placed until 12/9, not here yet). Try crosslinking beads to cells post fixation prior to staining and see if it doesn’t interfere with staining then and if beads remain stuck.

Formaldehyde cross-linking reading

  • quick overview:
  • Formaldehyde becomes methylene glycol in water, two hydroxyl groups
  • spectroscopic analysis of reaction of Gelatin with formaldehyde (Salsa et al 1996:) PC#1 saturates around 20-30 minutes.

first appearance of Lys-MeOH (PC#3), followed
by Arg-MeOH (PC#2) and by the Arg-Lys crosslink

Ph Project

  • Ajaz bringing new stained cells
  • Ph-647, Flag-cy7, for M and Wt
  • need to send figures

Wu lab collaboration

  • need to send figures to BB

Project 2

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