Daniel:Notebook/HiResChrPaint/2014-8-5
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No Wash Hybridization[edit]
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
- Allow stored samples to warm to room temperature
- Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
- Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
- Remove samples and allow to cool to room temperature
- Add 25 uL hybridization cocktail to center; sample C add 36 uL hybridization cocktail
- Add 22x22 #1.5 coverslip; seal with rubber cement
- Allow the rubber cement to air-dry for 5 minutes at room temperature
- Denature for 2.5 minutes at 92C; keep humid
- 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.
- Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
- For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
- VORTEX THESE SOLUTIONS VIGOROUSLY BEFORE USE EVERY TIME!
- Resuspend DNA in 20 uL labeling buffer (Component C)
- Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
- Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
- Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
- 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 |