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==Dye Couple== *Cot1 (4ug), and V6S2/V6S3 from [[Daniel:Notebook/HiResChrPaint/2014-6-30|yesterday]] #Dried samples in vacuum centrifuge #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) ## Resuspended V6S2 in 80 uL labeling buffer, split into 4 tubes #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 ##Added 1 uL 488 to each of Cot1 and V6S3 ##Added 5 uL 546 to each of the 4 V6S2 samples #Incubate at 80C for 25 minutes; stop the reaction by plunging the tube into an ice bath #Purify using a column (Centri-Sep recommended) ===Centri-Sep Column=== #Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel #Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle #Drain the column into a wash tube (200-250 uL will drain) #Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid) #Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface #Place column into collection tube and centrifuge at 750xg for 2 minutes #Dry sample in vacuum centrifuge ===Labeling Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#BFBFBF;font-size:12pt;font-weight:bold" align="center" valign="bottom" | width="65" height="30" | Sample | width="65" | ng/uL ssDNA | width="65" | pmol/uL dye | width="65" | pmol/uL ssDNA | width="65" | dye/probe | width="65" | bp:dye ratio |- style="font-size:12pt" align="center" valign="bottom" | height="15" | V6S2-546 | align="center" | 54.4 | align="center" | 2.2 | align="center" | 1.6 | align="center" | 1.4 | align="center" | 75 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | V6S3-488 | align="center" | 163.5 | align="center" | 0.7 | align="center" | 4.9 | align="center" | 0.1 | align="center" | 708 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Cot1-488 | align="center" | 50.3 | align="center" | 1 | align="center" | 0.5 | align="center" | 2.0 | align="center" | 152 |} Not great results, particularly for the 488 coupled reactions. I'm going to redo the labeling with the same sample, should enrich the 488 labeled DNA.
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