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==Amino Primers Test== ===qPCR=== 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="81" height="45" | | width="65" | 20 nM OligoPool | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AP1V41U | width="65" | 100 uM AP2V4 | width="65" | 100 uM AP1V61U | width="65" | 100 uM AP2V6 | width="65" | 2 mM aa-dUTP | width="65" | H20 | width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="15" | std 100 uL rxn | align="center" | 0.2 | align="center" | 50 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 2.5 | align="center" | 46.5 | align="center" | 100 |- style="font-size:12pt" align="center" | height="15" | V4S3* (2.5x) | align="center" | 0.5 | align="center" | 125 | align="center" | 1 | align="center" | 1 | align="center" | 0 | align="center" | 0 | align="center" | 0 | align="center" | 122.5 | align="center" | 250 |- style="font-size:12pt" align="center" | height="15" | V6S3* (2.5x) | align="center" | 0.5 | align="center" | 125 | align="center" | 0 | align="center" | 0 | align="center" | 1 | align="center" | 1 | align="center" | 0 | align="center" | 122.5 | align="center" | 250 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | V6S3 (2.5x) | align="center" | 0.5 | align="center" | 125 | align="center" | 0 | align="center" | 0 | align="center" | 1 | align="center" | 1 | align="center" | 6.25 | align="center" | 116.25 | align="center" | 250 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | V6S3- (2.5x) | align="center" | 0.5 | align="center" | 125 | align="center" | 0 | align="center" | 0 | align="center" | 1 | align="center" | 1 | align="center" | 0 | align="center" | 122.5 | align="center" | 250 |} 2. Aliquot 100 uL into each of 2 qPCR tubes (8 in total) 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii vii. 72C 2 min viii. 16C hold 4. Pool 2 samples together (200 uL each) 5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB 6. Measure in nanodrop ===Dye Coupling=== *Following [[Daniel:Protocols/AlexaDyeCouple|ARES Protocol]] 0. Dry samples in vacuum centrifuge 1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Add 5 uL DNA and 3 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 8 uL solvent; vortex β₯10 seconds *dye cannot be saved for later use. Use immediately! 5. Add 2 uL dye to each sample 6. Incubate in the dark for 1 hour 7. Add 15 uL nfH20 to sample 8. [[Daniel:Protocols/CentriSep|Centri-Sep purification]] 9. Measure in 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" | width="90" height="60" align="center" valign="bottom" | Sample | width="49" align="center" valign="bottom" | Post PCR ng/uL | width="48" align="center" valign="bottom" | Post PCR ug | width="56" align="center" valign="bottom" | Pre Spin ng/uL | width="78" align="center" valign="bottom" | Pre Spin ug | width="52" align="center" valign="bottom" | PreSpin pmol/uL ARES 546 | width="65" align="center" valign="bottom" | Post Spin ng/uL | width="77" align="center" valign="bottom" | Post Spin pmol/uL DNA | width="56" align="center" valign="bottom" | Post Spin ug | width="65" align="center" valign="bottom" | Post spin pmol/uL ARES 546 | width="65" align="center" valign="bottom" | Post Spin BP/dye | width="65" valign="bottom" | Dye/Probe |- style="font-size:12pt" |style="font-weight:bold" height="15" align="center" valign="bottom" | Am V4S3 | align="center" align="center" valign="bottom" | 55.5 | align="center" align="center" valign="bottom" | 2.2 | align="center" align="center" valign="bottom" | 12.4 | align="center" align="center" valign="bottom" | 6.2 | align="center" align="center" valign="bottom" | 11.4 | align="center" align="center" valign="bottom" | 51.4 | align="center" align="center" valign="bottom" | 0.8 | align="center" align="center" valign="bottom" | 1.3 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 312 | align="center" valign="bottom" | 0.7 |- style="font-size:12pt" |style="font-weight:bold" height="15" align="center" valign="bottom" | Am V6S3 | align="center" align="center" valign="bottom" | 63.3 | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 10.9 | align="center" align="center" valign="bottom" | 5.5 | align="center" align="center" valign="bottom" | 9.8 | align="center" align="center" valign="bottom" | 72 | align="center" align="center" valign="bottom" | 1.1 | align="center" align="center" valign="bottom" | 1.8 | align="center" align="center" valign="bottom" | 0.6 | align="center" align="center" valign="bottom" | 364 | align="center" valign="bottom" | 0.6 |- style="font-size:12pt" |style="font-weight:bold" height="15" align="center" valign="bottom" | V6S3 aa-dUTP | align="center" align="center" valign="bottom" | 57.1 | align="center" align="center" valign="bottom" | 2.3 | align="center" align="center" valign="bottom" | 12.7 | align="center" align="center" valign="bottom" | 6.4 | align="center" align="center" valign="bottom" | 11.4 | align="center" align="center" valign="bottom" | 58.4 | align="center" align="center" valign="bottom" | 0.9 | align="center" align="center" valign="bottom" | 1.5 | align="center" align="center" valign="bottom" | 0.6 | align="center" align="center" valign="bottom" | 295 | align="center" valign="bottom" | 0.7 |- style="font-size:12pt" |style="font-weight:bold" height="15" align="center" valign="bottom" | V6S3 | align="center" align="center" valign="bottom" | 61.9 | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 5.0 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 61.9 | align="center" align="center" valign="bottom" | 0.9 | align="center" align="center" valign="bottom" | 1.5 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1876 | align="center" valign="bottom" | 0.1 |} So it would appear that the amino primers do work. I'll have to do a formal run, however, before I can tell if they are truly as effective as using aa-dUTP. The main concern I have right now is that several aa-dUTP can be incorporated per probe, where as only 1 dye can be incorporated per probe with the primers. Therefore, the maximum dye/probe for amino modified primers is 1, but I have frequently had >1 (though close to 1) dye/probe with the aa-dUTP.
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