Daniel:Notebook/HiResChrPaint/2014-8-5: Difference between revisions
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>Djacobse (Created page with "=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...") |
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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. | 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== | ==Hybridization== | ||
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[[Category:HRCP]] [[Category:Hybridization]] [[Category:20140805]] | [[Category:HRCP]] [[Category:Hybridization]] [[Category:20140805]] | ||
=Cot1 Labeling (ULYSIS 488)= | |||
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) | |||
[[Image:ULYSIS_ReactionTable.png|500px]] |
Revision as of 21:23, 5 August 2014
No Wash Hybridization
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
- 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
- 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 37C or 42C
Cot1 Labeling (ULYSIS 488)
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