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=Linnarsson Lab Protocol 2= [[Daniel:Notebook/RNAFISH|Back to Calendar]] [[Daniel:Notebook/RNAFISH/2015-2-24|Last week]]'s RNA FISH protocol had too much autofluorescence (again), so I talked with Simone, who said paraformaldehyde was the biggest issue in reducing autofluorescence. He said to use freshly prepared solution, diluting it myself from 16% down to 4% and making it in PBS. Also, freezing it until ready to use. I've done all these things, so I am ready for another trial. Also, if this trial doesn't work, I can use the paraformaldehyde powder and make the whole thing myself. Finally, he said to avoid the green channel, so I will switch from using 488/594 to 546/647. ==Dye Coupling== *FOXP2-647/488 *GAD1-546/594 #Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature #Add 5 uL DNA and 3 uL of sodium bicarbonate buffer #Denature samples for 5 minutes at 95C, then snap cool using ice box #Dissolve 1 vial of reactive dye in 2 uL solvent; vortex β₯10 seconds #Add 2 uL dye to sample #Incubate in the dark for 1 hour #Add 10 uL 3M NaOAc and 80 uL nfH20 to sample #[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation ##Use TE Buffer #Ethanol Precipitation; incubate overnight #Continued [[Daniel:Notebook/RNAFISH/2015-3-5|tomorrow]] ==Buffer Prep== *Hybridization Buffer **RNAse free water 5.3 mL **SSC 20X 1 mL **Dextran sulfate 2 mL **Formamide 1 mL **E coli tRNA 500 uL **RVC 200 mM (warm to 37) 100 uL **BSA 50 mg/mL 40 uL
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