Daniel:Notebook/RNAFACS/2015-4-9
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Nuclei[edit]
Received nuclei from the Chun lab today. Based off Blue's protocol, I froze them down for future use.
Protocol[edit]
- Received nuclei in 15 mL eppendorf tubes containing ~1 mL of PBSE/BSA
- PBSE-PBS+ 2mM EGTA; BSA 1%
- Centrifuge in bucket rotor at 900xg for 10 min at 4C
- Remove supernatant and resuspend nuclei in 100 uL PBSE/BSA + 10% DMSO; pipette gently, do not use P10 or P2.5
- Optional step for filtration with 20 um filter; skipped this step
- Count Nuclei with cell counter
- Aliquot and freeeze down to -80C in styrofoam box or Mr. Frosty
- Store at -80C, use within a few months
- 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 |