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=Alu Control ([[Daniel:Notebook/HiResChrPaint/2014-1-8|Started 1/08/2014]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Dye Coupling Test== *Following [[Daniel:Protocols/AlexaDyeCouple#ARES|ARES Protocol]] '''Samples''' *[[Daniel:Notebook/HiResChrPaint/2014-1-9#Nanodrop Results|ALU2-C]]; total 1.0 ug *Human Cot-1 DNA, 1 ug 0. Vaccuum centrifuge DNA; resuspend in 2.5 uL nfH2O 1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Add 1.5 uL of sodium bicarbonate buffer 3. Denature samples for 5 minutes at 95C, then snap cool using ice box 4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex β₯10 seconds *dye cannot be saved for later use. Use immediately! 5. Add 1 uL dye to sample 6. Incubate in the dark for 1 hour 7. Add 20 uL nfH20 to sample; Measure concentration in Nanodrop 8. [[Daniel:Protocols/CentriSep|Centri-Sep column purification]] 9. Measure concentration in Nanodrop ===Labeling Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="30" | Sample | width="65" | ng/uL DNA | width="65" | pmol/uL dye | width="71" | pmol/uL DNA | width="71" | dye/probe | width="71" | Base:Dye Ratio (:1) | width="65" | DNA Capture % | width="65" | Dye Capture % |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | Alu2C-Pre Column | align="center" | 588.4 | align="center" | 130.7 | align="center" | 8.9 | align="center" | 14.7 | align="center" | 14 | N/A | N/A |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | Cot1-Pre Column | align="center" | 527.8 | align="center" | 136.6 | align="center" | 8.0 | align="center" | 17.1 | align="center" | 12 | N/A | N/A |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | Alu2C-Post Column | align="center" | 18 | align="center" | 0.1 | align="center" | 0.3 | align="center" | 0.4 | align="center" | 545 | align="center" | 3.82 | align="center" | 0.10 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | Cot1-Post Column | align="center" | 30.2 | align="center" | 2.8 | align="center" | 0.5 | align="center" | 6.1 | align="center" | 33 | align="center" | 7.15 | align="center" | 2.56 |} So just like the results from [[Daniel:Notebook/HiResChrPaint/2014-1-10#Labeling Results|Friday]], the Cot1 DNA seems to have much better results than the Alu DNA. Since the Cot1 DNA has no ALU elements there should be no reason why the Cot1 DNA should be coupling at all. ==qPCR== In order to better assess the dye coupling, I'm going to repeat the experiment but also add in a positive control. For that I'll use V6S3, my favorite probe set to amplify. V6S3 has been shown to amplify well in the past and has good labeling results. {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" |style="background-color:#FFFF00" align="center" width="65" height="45" | | width="65" | 10 nM OligoPool | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AP1V61U-Amino | width="65" | 100 uM AP2V6 | width="65" | H20 | width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="15" | per rxn | align="center" | 0.4 | align="center" | 50 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 48.8 | align="center" | 100 |- style="font-size:12pt" align="center" | height="15" | V6S3 (6.1x) | align="center" | 2.44 | align="center" | 305 | align="center" | 2.44 | align="center" | 2.44 | align="center" | 297.68 | align="center" | 610 |} ===qPCR Results=== [[Image:2014-01-13-V6S3.png|500px]] ===Nanodrop Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="15" | Sample | width="65" | ng/uL | width="65" | ug in 40 uL | width="65" | ug total |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | V6S3A | align="center" | 73.6 | align="center" | 2.9 | align="center" | |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | V6S3B | align="center" | 70.4 | align="center" | 2.8 | align="center" | 5.8 |}
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