Daniel:Notebook/HiResChrPaint/2014-8-5

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Revision as of 16:12, 7 August 2014 by >Djacobse (→‎Hybridization)
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No Wash Hybridization[edit]

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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[edit]

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; sample C add 36 uL hybridization cocktail
  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 42C

Cot1 Labeling (ULYSIS 488)[edit]

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[edit]

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