Friday 11/22/13

9:45a – 11:50a
(remotely)

12:00p – 7:10p

Genomics

  • A549 whole cell data finished running cufflinks. Bottom half of genes are empty / all have zero FKPM (chr12 on).
  • attempting to run cufflinks 2.1 centos6 version on cytosol BAM file directly
  • Run direct on BAM fails.
  • SAM files too big to run from home scratch directory. Moving to Zhuang lab directory.

RC computing

  • WinSCP onto login.rc.fas.harvard.edu
  • cd /n/nss2b/scratch/scratch08/zhuang_lab/
  • calculating size of zhuang_lab (max is 1Tb, hopefully there is enough space here to convert all the BAM to SAM so we can run cufflinks even though the header files from ENCODE are crap).
  • Yari’s folder here is empty and mine has only 12 Gb, (now more like 100), so we should be fine without blowing this cap yet.
  • Or we could if WinSCP / odyssey connection wouldn’t keep on dropping me. This is very annoying.
  • WinSCP is EXTREMELY (impossibly?) slow at deleting large numbers of files from server (e.g. 3 entries per gene in the human genome). Connection stays at 0% deleting forever and then hangs.
  • SAM2BAM on A549 NODE_FAIL. what’s this mean?

Ph project

  • no effect of PhM concentration at all in koff model. Is this a perfectly balanced composition? Weaker binding but higher conc?
  • no, I think it’s just the capping model is effective at quenching binding at pretty low concentrations
  • reality is probably a hybrid condition, a bit weaker association as well as lower

STORM2 alignment

  • if 1st two objective mirrors won’t align optics, use third mirror up down.
  • Realigned back optics
  • Reset back apeture for coarse alignment so it is in register with the aligned beam (previously was not).
  • Replaced missing screw on Quadview
  • Realigned Quad view following directions
  • STORM2 and quadview now algined
  • imaged beads
  • imaged negative control for D12, looks excellent. Imaged with crosslinked beads. Beads much too sparse. Try 1:500 next time?
  • Etching works best with VERY LIGHT TOUCH! deeply scoring gives ugly marks AND causes coverslip to break! Tragedy!
  • D12 RNA sample destroyed. Try again tomorrow with E10 sample

STORM analysis

  • G9 data now running on Tuck (should have done this earlier!)
  • G1 data now running splitdax (and conflicting badly with the disk write to the G9 data, but oh well).

Cell culture

  • Cells growing excellently.
  • Main original 3 flasks will need passage again tomorrow — move to large flask, grow up densly in prep for freezing.
  • Split off separate culture flask for Bogdan with separate stock solution. Need to order new stock vial tomorrow.
  • Plated and fixed 12 coverslips of cells for Bogdan and myself each.
  • Take these through the prep protocol tomorrow.
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