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===Day 3=== #Wash with 1X PBS once #To DARTFISH slide add 1uM FISSEQ_ppRCA (2ul 200uM FISSEQ_ppRCA + 398ul 2X SSC + 30% formamide) and incubate 1hr at 55C #*'''DARTFISH slide was dried out''' #*'''Edit: the slide may not have dried out so much as leaked. The slide has been leaking since this step.''' #**Possible the 3M 200M adhesive is not resistant to long term high temp incubation. #To FISSEQ slide and dish add 1uM FISSEQ_RCA (6ul 100uM FISSEQ_RCA + 594ul 2X SSC + 30% formamide) and incubate 1hr at 55C (should be 60C but only one oven) #*Preheated to 70C before adding #Wash with 2X SSC once, 1X SSC once, and 1X PBS once #Add RCA mix and incubate 6hr at 30C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||174 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | Phi 29 (low conc)||2 |- | Total||200 |} #Wash with 1X PBS once #Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate at RT for 1hr #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate at RT for 30min #Wash with 1X PBS twice and store in 1X PBS at 4C
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