Daniel:Notebook/RNAFACS/2015-4-9

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Nuclei[edit]

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

Protocol[edit]

  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[edit]

Species Nuclei Count (#/mL) Volume (uL) Total Nuclei Received Volume of Aliquots (uL) Number of Aliquots Nuclei/Aliquot
Mouse 5.36E+06 100 5.36E+05 10 10 5.36E+04
Human 1.75E+07 500 8.75E+06 30 15 5.25E+05