Daniel:Notebook/HiResChrPaint/2014-8-5: Difference between revisions

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|- style="background-color:#8DB4E2;font-size:12pt" align="center"
|- style="background-color:#8DB4E2;font-size:12pt" align="center"
|style="font-weight:bold" width="65" height="30" | Sample
|style="font-weight:bold" width="65" height="30" | Sample
|style=" width="65" |  Cot1-488 (below) (ug)
|style="font-weight:bold" width="65" |  Cot1-488 (below) (ug)
|style="font-weight:bold;text-decoration:none" width="65" | Dystrophin-488 (pmol)
|style="font-weight:bold;text-decoration:none" width="65" | Dystrophin-488 (pmol)
|style="font-weight:bold" width="65" | Dystrophin-546 (pmol)
|style="font-weight:bold" width="65" | Dystrophin-546 (pmol)

Revision as of 16:06, 6 August 2014

No Wash Hybridization

Back to Calendar

I'll be continuing my FISH experiments with a modified protocol. I'm going to skip the wash steps, fearing that most probe is being washed away during these phases. Alternatively, I'm going to explore a change in the protocol. I found in a more in depth Beliveau publication that the oligos are not resuspended, for instance after vacuum centrifuge like I have been doing, but are rather added in a concentrated fashion to the hybridization mix, usually ≤4 uL.


Hybridization

Sample Matrix

Sample Cot1-488 (below) (ug) Dystrophin-488 (pmol) Dystrophin-546 (pmol) Cot1 (ug)
A 0 8.6 (30) 0 2.5 (2.5)
B 0 0 7.9 (30) 2.5 (2.5)
C* 0 5 (17.5) 0 2.5 (2.5)
D 8.5 (0.4) 0 0 0
  • denotes this sample was not dried using vacuum centrifuge
  1. Allow stored samples to warm to room temperature
  2. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
  3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
  4. Remove samples and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to center
  6. Add 22x22 #1.5 coverslip; seal with rubber cement
  7. Allow the rubber cement to air-dry for 5 minutes at room temperature
  8. Denature for 2.5 minutes at 92C; keep humid
  9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C

Cot1 Labeling (ULYSIS 488)

Ran out of positive control so I'm labeling another.

  1. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
    1. For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
    2. VORTEX THESE SOLUTIONS VIGOROUSLY BEFORE USE EVERY TIME!
  2. Resuspend DNA in 20 uL labeling buffer (Component C)
  3. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
  4. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
  5. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
  6. Purify using a column (Centri-Sep recommended)
File:ULYSIS ReactionTable.png

Labeling Results

Sample ng/uL DNA pmol/uL dye pmol/uL DNA base:dye
Cot1-488-A 47.3 0.9 0.5 80
Cot1-488-B 51.3 0.8 0.5 97