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=In Tube FISH Test (Started [[Daniel:Notebook/RNAFACS/2015-4-10|4/10/2015]]) = [[Daniel:Notebook/RNAFACS|Back to Calendar]] ==Dye Labeling== #Centrifuge at 4 C for 30 minutes (14000 rpm) #Remove supernatant and add 750 uL chilled 75% EtOH #Centrifuge at minutes at 4 C #Dry the pellet in the hood #Resuspend pellet in 12 uL TE ===Labeling Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" | width="96" height="30" valign="bottom" | Probe | width="65" align="center" | ng/uL ssDNA | width="65" align="center" | pmol/uL dye | width="85" align="center" | total DNA (ug) | width="65" align="center" | dye/probe | width="65" align="center" | bp:dye ratio | width="85" align="center" | DNA Yield (%) |- style="font-size:12pt" | height="15" valign="bottom" | GAD1-488 | align="center" align="center" valign="bottom" | 96.4 | align="center" align="center" valign="bottom" | 16.0 | align="center" align="center" valign="bottom" | 1.4 | align="center" align="center" valign="bottom" | 1.1 | align="center" align="center" valign="bottom" | 18 | align="center" align="center" valign="bottom" | 64.84 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | RBFOX3-546 | align="center" align="center" valign="bottom" | 70.0 | align="center" align="center" valign="bottom" | 10.5 | align="center" align="center" valign="bottom" | 1.1 | align="center" align="center" valign="bottom" | 1.0 | align="center" align="center" valign="bottom" | 20 | align="center" align="center" valign="bottom" | 50.00 |- style="font-size:12pt" | height="15" valign="bottom" | FOXP2-594 | align="center" align="center" valign="bottom" | 30.3 | align="center" align="center" valign="bottom" | 5.5 | align="center" align="center" valign="bottom" | 1.1 | align="center" align="center" valign="bottom" | 1.2 | align="center" align="center" valign="bottom" | 17 | align="center" align="center" valign="bottom" | 21.87 |} ==Hybridization== #Pellet nuclei at 1000xg at 4C for 5 minutes; remove supernatant #Resuspend nuclei in 150 uL RPHB #Split nuclei into 3 tubes (50 uL each; ~150K nuclei/tube) #Add 1 uL labeled probes to 0.5-1ng/uL #Incubate overnight at 30C
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