Monday 08/17/15

9:45 am

RNA library prep

setup

  • protocol from NEB
  • discuss plans with Jeff
  • request training on BioAnalyzer and/or TapeStation instrument for library QC
  • collecting kit components

RNA purification

  • run out 1 uL on a 1% agarose gel in TAE
  • treat samples with NEB DNase I (1 uL in 20 uL reaction, used RQ1 DNase buffer, don’t have an NEB one. validated the RQ1 is also a DNase I from Promega, guaranteed RNase free)
  • Purify samples on AMPure RNA XP beads.
  • performed all washes on column
  • gel results: (clearly degraded). RNA is a month old (stored at -80C, but used for several RTPCR runs.
    degraded_TotRNA
  • probably do better to start 1 clean fresh run with newly isolated samples.
  • we can run through the procedure today with these samples to get familiar with the protocol.

First strand synthesis

  • ran without actinomyocin (don’t have any). Already did DNA digestion so hopefully this isn’t necessary

Second strand synthesis

  • setup is easy
  • run bead cleanup using Aline beads from Jeff in place of AMPure XP beads

More steps

  • purify dsDNA (beads)
  • end prep
  • adapter ligation
  • purify DNA (beads)
    • this uses a lot of beads. Ordered more Aline beads from Aline biosciences
  • PCR enrichment
    • ran on QPCR machine with EvaGreen
    • 14 cycles, started to saturate, pulled off
    • testing sample by running out product on a gel
    • Typhoon isn’t working — images are perfectly 100% blank. no noise, no contrast, no background of gel.
  • still have 1 more bead clean up to do.

STORM analysis

  • compute z-calibration from surface beads for STORM2 for Ella
  • cancelled STORM imaging for tonight (insufficient time to setup a run before midnight, insufficient time to collect 2 color data if the staining works before 10am).
  • signed up for STORM4 for Thursday and Friday for live imaging with Steven. STORM4 might not work — the 100x objective went missing.

Ph KD immuno

  • finished staining
  • tested samples on Turnkey. WT shows decent staining. Ph in KD not detectable, but need nuclear counterstain to validate cell region.
  • maybe try imaging these on confocal tomorrow.

Editing

  • Editing recommendations for YF application
  • Editing Ph manuscript draft
  • got new Ph manuscript draft, will try to add to current one.
This entry was posted in Summaries. Bookmark the permalink.