Daniel:Notebook/RNAFACS/2015-4-9: Difference between revisions
Jump to navigation
Jump to search
>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== #...") |
>Djacobse |
||
Line 18: | Line 18: | ||
===Nuclei Counting and Aliquots=== | ===Nuclei Counting and Aliquots=== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B7DEE8;font-size:12pt" align="center" | |||
| width="65" height="30" | Species | |||
| width="81" | Nuclei Count (#/mL) | |||
| width="65" | Volume (uL) | |||
| width="65" | Total Nuclei Received | |||
| width="82" | Volume of Aliquots (uL) | |||
| width="65" | Number of Aliquots | |||
| width="77" | Nuclei/Aliquot | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | Mouse | |||
| align="center" | 5.36E+06 | |||
| align="center" | 100 | |||
| align="center" | 5.36E+05 | |||
| align="center" | 10 | |||
| align="center" | 10 | |||
| align="center" | 5.36E+04 | |||
|- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | |||
| height="15" | Human | |||
| align="center" | 1.75E+07 | |||
| align="center" | 500 | |||
| align="center" | 8.75E+06 | |||
| align="center" | 30 | |||
| align="center" | 15 | |||
| align="center" | 5.25E+05 | |||
|} |
Latest revision as of 18:16, 10 April 2015
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 |