Tuesday 07/07/15

Posted on July 7, 2015 by admin

9:00 am – 12:00 am

Goals

  • MERFISH report requested by XZ. try to get L15 data analyzed for Wed
  • Chromatin report requested by XZ. put together slides for this meeting
  • finish slides for SDB meeting
  • proof-read DF application and prepare to mail.
  • Plate and fix RNAi knockdown cells (5 pm).

Cell culture

  • BG3 cells arrived
    • lots of cells: moved to 75cm2 flask + 2 small 25cm2 flask after the settled
    • need insulin for growth media. Ordered this today, hopefully it arrives soon.
  • old Hs Kc cells too dense.
  • Plated new Kc cells. allowed 1 hr to attach and settle and then Heat Shocked for 2 hours at ~37C.

Knockdown experiments

prepped RNA

  • sample order
    1. mock
    2. PPES
    3. Psc
    4. Ph-p, Ph-d
    5. Pho
    6. Su(z)2
  • extracted RNA, using just 1.5 mL of cells and Qiagen Midi kit (350 uL lysis buffer)
  • concentrations in the 100-300 ng/uL range

plated cells

  • 500 uL per coverglass of cells. Reasonable confluence, get 6 coverslips + 1 mL for RNA extraction

HS experiments

  • 2 hour heat shock treatment (see above)
  • isolated / prepped RNA
  • concentration in the 500 – 700 ng/uL range

MERFISH

  • found bug in previous normalization (was ID’ing TCEP movies wrong)
  • fixed normalization
  • default decoding still doesn’t work.

Presentation

  • proof-reading and editing DF app
  • working on slides for SDB
  • contacting Levine lab members for get together
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