Daniel:Notebook/RNAFACS/2015-4-10: Difference between revisions

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>Djacobse
(Created page with "=In Tube FISH Test= ==Nuclei Fixation and Dehydration== #Fix cells in 4% paraformaldehyde for 5 min #Centrifuge cells at 1000g for 5 minutes #Wash cells with 70% EtOH #Ethan...")
 
>Djacobse
 
(3 intermediate revisions by the same user not shown)
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=In Tube FISH Test=
=In Tube FISH Test=
[[Daniel:Notebook/RNAFACS|Back to Calendar]]


==Nuclei Fixation and Dehydration==
==Nuclei Fixation and Dehydration==
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##Use TE Buffer
##Use TE Buffer
#Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
#Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
#Continued [[Daniel:Notebook/RNAFACS/2015-4-9|tomorrow]]
#Continued [[Daniel:Notebook/RNAFACS/2015-4-11|tomorrow]]
 
===Spin Column Results===
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CCC0DA;font-size:12pt"
| width="96" height="30"  valign="bottom" | Probe
| width="65" align="center" | ng/uL ssDNA
| width="65" align="center" | pmol/uL dye
| width="85" align="center" | total DNA (ug)
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | GAD1-488
| align="center" align="center" valign="bottom" | 22.3
| align="center" align="center" valign="bottom" | 4.4
| align="center" align="center" valign="bottom" | 2.23
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | RBFOX3-546
| align="center" align="center" valign="bottom" | 21.0
| align="center" align="center" valign="bottom" | 13.0
| align="center" align="center" valign="bottom" | 2.1
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | FOXP2-594
| align="center" align="center" valign="bottom" | 48.5
| align="center" align="center" valign="bottom" | 33.5
| align="center" align="center" valign="bottom" | 4.85
 
|}

Latest revision as of 23:46, 4 June 2015

In Tube FISH Test[edit]

Back to Calendar

Nuclei Fixation and Dehydration[edit]

  1. Fix cells in 4% paraformaldehyde for 5 min
  2. Centrifuge cells at 1000g for 5 minutes
  3. Wash cells with 70% EtOH
  4. Ethanol permeabilization- 70% EtOH overnight at 4C

Dye Labeling[edit]

  • GAD1(48)-488
  • FOXP2(48)-594
  • RBFOX3(24)-546
  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 8 uL sample to dye tube
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL nfH20 to sample
  8. Centri-Sep column purification after incubation
    1. Use TE Buffer
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
  10. Continued tomorrow

Spin Column Results[edit]

Probe ng/uL ssDNA pmol/uL dye total DNA (ug)
GAD1-488 22.3 4.4 2.23
RBFOX3-546 21.0 13.0 2.1
FOXP2-594 48.5 33.5 4.85