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==ULYSIS Labeling== I'm going to label the padlock probes and some Cot1 with ULYSIS dye kits for FISH. I'll label the padlock probes with Alexa 488 and the Cot1 DNA with Alexa 546. '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt" align="center" | width="65" height="35" | Sample | width="65" | ng/uL | width="65" | uL added | width="65" | ug input | width="65" | Dye | width="65" | uL dye |- style="font-size:12pt" | height="15" valign="bottom" | PPRL-A1 | align="center" align="center" valign="bottom" | 154.1 | align="center" align="center" valign="bottom" | 40 |style="font-weight:bold" align="center" align="center" valign="bottom" | 6.2 | align="center" valign="bottom" | Alexa-488 | align="center" align="center" valign="bottom" | 5 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | PPRL-A2 | align="center" align="center" valign="bottom" | 131 | align="center" align="center" valign="bottom" | 40 |style="font-weight:bold" align="center" align="center" valign="bottom" | 5.2 | align="center" valign="bottom" | Alexa-488 | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" | height="15" valign="bottom" | Cot1-A | align="center" align="center" valign="bottom" | 1000 | align="center" align="center" valign="bottom" | 5 |style="font-weight:bold" align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | Alexa-546 | align="center" align="center" valign="bottom" | 5 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Cot1-B | align="center" align="center" valign="bottom" | 1000 | align="center" align="center" valign="bottom" | 5 |style="font-weight:bold" align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | Alexa-546 | align="center" align="center" valign="bottom" | 5 |} #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|thumb]] ===Labeling Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#E6B8B7;font-size:12pt" align="center" | width="65" height="30" | Sample | width="68" | ng/uL dsDNA | width="65" | pmol/uL dye | width="66" | pmol/uL dsDNA | width="71" | pmol probe in 50 uL | width="66" | dye/probe | width="65" | bp:dye |- style="font-size:12pt" | height="15" valign="bottom" | PPRL-A1 | align="center" align="center" valign="bottom" | 92.6 | align="center" align="center" valign="bottom" | 2.7 | align="center" align="center" valign="bottom" | 1.4 | align="center" align="center" valign="bottom" | 70.2 | align="center" align="center" valign="bottom" | 1.9 |style="font-weight:bold" align="center" align="center" valign="bottom" | 52 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | PPRL-A2 | align="center" align="center" valign="bottom" | 93.8 | align="center" align="center" valign="bottom" | 2.8 | align="center" align="center" valign="bottom" | 1.4 | align="center" align="center" valign="bottom" | 71.1 | align="center" align="center" valign="bottom" | 2.0 |style="font-weight:bold" align="center" align="center" valign="bottom" | 51 |- style="font-size:12pt" | height="15" valign="bottom" | Cot1-A | align="center" align="center" valign="bottom" | 47.7 | align="center" align="center" valign="bottom" | 1.1 | align="center" align="center" valign="bottom" | 0.4 | align="center" align="center" valign="bottom" | 18.1 | align="center" align="center" valign="bottom" | 3.0 |style="font-weight:bold" align="center" align="center" valign="bottom" | 66 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Cot1-B | align="center" align="center" valign="bottom" | 50.7 | align="center" align="center" valign="bottom" | 1.1 | align="center" align="center" valign="bottom" | 0.4 | align="center" align="center" valign="bottom" | 19.2 | align="center" align="center" valign="bottom" | 2.9 |style="font-weight:bold" align="center" align="center" valign="bottom" | 70 |}
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