Daniel:Notebook/RNAFACS/2015-4-9

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Revision as of 18:11, 10 April 2015 by >Djacobse (Created page with "=Nuclei= Back to Calendar Received nuclei from the Chun lab today. Based off Blue's protocol, I froze them down for future use. ==Protocol== #...")
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Nuclei

Back to Calendar

Received nuclei from the Chun lab today. Based off Blue's protocol, I froze them down for future use.

Protocol

  1. Received nuclei in 15 mL eppendorf tubes containing ~1 mL of PBSE/BSA
    1. PBSE-PBS+ 2mM EGTA; BSA 1%
  2. Centrifuge in bucket rotor at 900xg for 10 min at 4C
  3. Remove supernatant and resuspend nuclei in 100 uL PBSE/BSA + 10% DMSO; pipette gently, do not use P10 or P2.5
    1. Optional step for filtration with 20 um filter; skipped this step
  4. Count Nuclei with cell counter
  5. Aliquot and freeeze down to -80C in styrofoam box or Mr. Frosty
    1. Store at -80C, use within a few months
    2. When thawing, thaw rapidly in hand, gently pipette 6-8 times using P20 and add reagent quickly

Nuclei Counting and Aliquots