Daniel:Notebook/RNAFISH/2015-3-5: Difference between revisions
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>Djacobse (Created page with "=Linnarsson Lab Protocol 2 (Started 3/4/2015)= Back to Calendar ==Ethanol Precipitation== #Remove from -80C...") |
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[[Daniel:Notebook/RNAFISH|Back to Calendar]] | [[Daniel:Notebook/RNAFISH|Back to Calendar]] | ||
==Ethanol Precipitation== | ==Dye Coupling (Continued; Ethanol Precipitation)== | ||
#Remove from -80C | #Remove from -80C | ||
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===Nanodrop Results=== | ===Nanodrop Results=== | ||
==Hybridization== | ==Hybridization== |
Revision as of 17:27, 5 March 2015
Linnarsson Lab Protocol 2 (Started 3/4/2015)
Dye Coupling (Continued; Ethanol Precipitation)
- Remove from -80C
- Centrifuge at 4 C at 12000 rpm for 30 minutes
- Remove supernatant and add 700 uL chilled 75% EtOH
- Centrifuge at minutes at 4 C for 10 minutes at 12000 rpm
- Remove supernatant and dry the pellet in the hood
- Resuspend pellet in 7.5 uL TE buffer
- Store DNA at 4C for immediate use
Nanodrop Results
Hybridization
Sample Matrix
- Prepare fresh paraformaldehyde 4% and keep at RT
- Fix sections with 4% paraformaldehyde for 15 minutes at RT
- Rinse sections twice with RT PBS
- Permeabilization-may use:
- Ethanol (70% EtOH, >90 minute incuabation at 4C)
- Rinse sections twice with RT PBS
- Prepare the wash buffer and let it sit at RT
- Warm hybridization buffer and warm it up to 37C
- Prepare hybridization reaction:
- hybridization buffer-100 uL; probe 2-10 uL
- Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.