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=Alu Control ([[Daniel:Notebook/HiResChrPaint/2014-1-8|Started 1/08/2014]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Production qPCR== Since I didn't make enough the [[Daniel:Notebook/HiResChrPaint/2014-1-8#Production qPCR|first time]], I'm going to do another production PCR to make more Alu probe. 1. Reaction Table {| 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" | |style="background-color:#D9D9D9;font-weight:bold" width="65" | gDNA |style="background-color:#D9D9D9;font-weight:bold" width="65" | 2X Kapa SYBR qPCR MM |style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP1V6-Am |style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP2V6 |style="background-color:#D9D9D9;font-weight:bold" width="65" | H20 |style="background-color:#D9D9D9;font-weight:bold" width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="15" | per rxn | align="center" | 1 | align="center" | 50 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 48 | align="center" | 100 |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="30" | PGP1F (12.1X) | align="center" | 12.1 | align="center" | 605 | align="center" | 6.05 | align="center" | 6.05 | align="center" | 580.8 | align="center" | 1210 |} 2. Aliquot 100 uL into each of 3 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 10 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x30 vii. 72C 2 min viii. 16C hold 4. Pool 3 samples together each 5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB 6. Measure concentrations in nanodrop ===qPCR Results=== [[Image:2014-01-10-AluProduction.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" | ALU3-A | width="65" | ALU3-B | width="65" | ALU3-C | width="65" | ALU3-D |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 69.6 | align="center" | 70.3 | align="center" | 69.9 | align="center" | 56 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 2.8 | align="center" | 2.8 | align="center" | 2.8 | align="center" | 2.2 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total | align="center" | | align="center" | | align="center" | | align="center" | 10.6 |} Much better amplification results this time. Next I'll check dye coupling results. ==Dye Coupling Test== *Following [[Daniel:Protocols/AlexaDyeCouple#ARES|ARES Protocol]] '''Samples''' *[[Daniel:Notebook/HiResChrPaint/2014-1-9#Nanodrop Results|ALU2-AB]]; total 1.1 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" | Alu-Pre Column | align="center" | 460 | align="center" | 63 | align="center" | 7.0 | align="center" | 9.0 | align="center" | 22 | N/A | N/A |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | Cot1-Pre Column | align="center" | 44.7 | align="center" | 43.9 | align="center" | 0.7 | align="center" | 64.8 | align="center" | 3 | N/A | N/A |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | Alu-Post Column | align="center" | 26.6 | align="center" | 1.3 | align="center" | 0.4 | align="center" | 3.2 | align="center" | 62 | align="center" | 7.23 | align="center" | 2.58 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | Cot1-Post Column | align="center" | 33.3 | align="center" | 4.7 | align="center" | 0.5 | align="center" | 9.3 | align="center" | 21 | align="center" | 93.12 | align="center" | 13.38 |}
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