Wednesday 04/09/14

9:30a – 8:30p

Chromatin labeling

  • stains failed again. No stains yet detected with this batch of cells.
    • maybe it’s a cell prep issue? This F07 probe worked fine before.
    • maybe some reagents went bad in the cell prep? Make new formamide and hybe buffer

Probe Making

qPCR followup

  • Run on new 2% gel 2.5 uL of the second elution of DNA from the limited-cycle PCR
  • Melted gel. Should remember to always change 100% of the buffer and let’s go back to 10 instead of 11 min.
  • lanes: Lib3 E05-E12 (top), F01-F08 (bottom). Also showing yesterdays gel on old gel (not a good week for gels!)

E5-E12_F1-F8 melted_qPCRgel

RT reactions

  • individual reactions: 10 uL RNA + 4 uL primer + 4 uL RT buffer + 2 uL dNTPs + 1 uL inhibitor + .1 uL
  • all prepared with P1 primer
  • Master mix: 18*4 uL primer = 72 uL
  • 72 uL primer + 72 uL RT buffer + 36 uL dNTPs + 6 uL Maxima enzyme + 18 uL inhibitor. 10 uL each, add to 10 uL RNA.
  • out of dNTPs, thawing new dNTPs
  • running RT reactions
  • using last 2 pre-cast gels. Ordered more gels today.
    • also ordered more T7 high yield quick mix kit
    • more DCC-5 columns


Troubleshooting Cajal Server 2012 Insight compatability issues

  • InsightM gives “Frame out of range!” pop-up error when passed region parameters on Cajal windows 2012.
  • This error requires the user to press okay. There-after it seems to analyze just fine. This includes processing the ROI correctly. Batch analysis is suspended until the OK is pressed — very disruptive to automated processing.
  • copied files to Monet, run fine using the exact same InsightM version.
  • wrote to Bo to comment on bug.
  • Meanwhile commented out the ROI parameters on system run in Cajal local.

Cell staining

  • passaged cells
  • created new 75 cm^2 flask of cells at mid-density for Bogdan.
  • plated cells — reprep new cells for hybes. See if a possible cell prep issue is why my hybes suddenly started failing?
  • New Hybe Dilution Mix, 10 mL, 1.4X
    • 2.8 mL 50% dextran sulfate
    • 1.4 mL 2X SSC
    • 6.5 mL DI formamide
    • 100 uL 10% Tween 20
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