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=Alu Control ([[Daniel:Notebook/HiResChrPaint/2014-1-8|Started 01/08/2014]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Cot1 Labeling== Labeling lots of Cot1 DNA for FISH experiments. '''Samples''': *5 ug Cot1 DNA *The entire sample of [[Daniel:Notebook/HiResChrPaint/2014-1-13#Nanodrop Results|V6S3-B]] ===Protocol=== *[[Daniel:Protocols/AlexaDyeCouple#ULYSIS|Following ULYSIS protocol]] 1. 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'''! 2. Resuspend DNA in 20 uL labeling buffer (Component C) 3. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly 4. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL 5. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath 5-1 Measure pre-column concentration using nanodrop 6. Purify using a [[Daniel:Protocols/CentriSep|Centri-Sep column]] ===Labeling Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" valign="bottom" | align="center" width="65" height="15" | |style="background-color:#8DB4E2;font-weight:bold" width="65" colspan="4" | Pre Column |style="background-color:#E6B8B7;font-weight:bold" width="65" colspan="4" | Post Column | align="center" width="71" | | align="center" width="65" | |- style="font-size:12pt;font-weight:bold" align="center" |style="background-color:#D9D9D9" height="45" | Sample |style="background-color:#8DB4E2" | ng/uL ssDNA |style="background-color:#8DB4E2" | pmol/uL dye |style="background-color:#8DB4E2" | pmol/uL ss probe |style="background-color:#8DB4E2" | base:dye |style="background-color:#E6B8B7" | ng/uL ssDNA |style="background-color:#E6B8B7" | pmol/uL dye |style="background-color:#E6B8B7" | pmol/uL ss probe |style="background-color:#E6B8B7" | base:dye |style="background-color:#D8E4BC" | DNA retention (%) |style="background-color:#D8E4BC" | Dye retention (%) |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="15" | Cot1 |style="background-color:#8DB4E2" align="center" | 145.2 |style="background-color:#8DB4E2" align="center" | 36.7 |style="background-color:#8DB4E2" align="center" | 2.5 |style="background-color:#8DB4E2" align="center" | 12 |style="background-color:#E6B8B7" align="center" | 80.5 |style="background-color:#E6B8B7" align="center" | 4.6 |style="background-color:#E6B8B7" align="center" | 1.4 |style="background-color:#E6B8B7" align="center" | 53 |style="background-color:#D8E4BC" align="center" | 44.35 |style="background-color:#D8E4BC" align="center" | 10.03 |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="15" | V6S3 |style="background-color:#8DB4E2" align="center" | 76.4 |style="background-color:#8DB4E2" align="center" | 24.6 |style="background-color:#8DB4E2" align="center" | 2.9 |style="background-color:#8DB4E2" align="center" | 9 |style="background-color:#E6B8B7" align="center" | 57.7 |style="background-color:#E6B8B7" align="center" | 0.9 |style="background-color:#E6B8B7" align="center" | 2.2 |style="background-color:#E6B8B7" align="center" | 194 |style="background-color:#D8E4BC" align="center" | 60.42 |style="background-color:#D8E4BC" align="center" | 2.93 |} These are great results, and I now have plenty of DNA for FISH.
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