Daniel:Notebook/RNAFACS/2015-4-9: Difference between revisions

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(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
 
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===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]

Back to Calendar

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