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=Primer24 qPCR Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== This tests the off-target binding present. Since I don't have primer24 qPCR primer (yet, just ordered) I can only repeat the [[Daniel:Notebook/ComboLock/2016-9-29|AmpR-primer12RC]] test that I did on 9/29/16. The expected product is again 157bp. <ol> <li>qPCR</li> <ol type="A"> <li>Set up qPCR reactions according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" | width="105" height="45" | Sample | width="65" | Molecule Count | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="105" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM primer12RC | width="65" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="65" | Total Volume (uL) |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 0A | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | A1 | align="center" align="center" valign="bottom" | 37 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 0B | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | A2 | align="center" align="center" valign="bottom" | 37 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 0C | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | A3 | align="center" align="center" valign="bottom" | 37 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC (rev) | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | A4 | align="center" align="center" valign="bottom" | 37 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 19.5 | align="center" align="center" valign="bottom" | 50 |} <li>Make the following master mix</li> <ol type="a"> <li>71.75 uL nfH2O</li> <li>2.05 uL 100 uM primer12 RC</li> <li>20.5 uL 10 uM Amp6.3R Index 30</li> <li>102.5 uL Kapa SYBR Fast</li></ol> <li>Aliquot 43 uL MM1 into each well</li> <li>Add 2 uL sample to appropriate well</li> <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x26</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 48 uL TBE, 12 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 22 minutes at 250V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20161001-qPCR-LatchX4-Primer12RC.png|qPCR Curve File:2016-10-03-PCAmp4-Primer12RC.png|Gel Image </gallery> ==Primer Concentration Test== The primer concentrations might be high. To test I'm doing dilutions, reminiscent of [[Daniel:Notebook/ComboLock/2016-9-26|Monday, 9/26/2016]]. <ol> <li>qPCR</li> <ol type="A"> <li>Set up qPCR reactions according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" | width="105" height="45" | Sample | width="65" | Molecule Count | width="65" | Lane | width="65" | AmpR Index | width="65" | Sample Vol (uL) | width="105" | 2X Kapa SYBR qPCR MM | width="65" | 10 uM AmpF 6.4 | width="65" | 10 uM AmpR6.3-IndXX | width="65" | H2O | width="65" | Total Volume (uL) | width="65" | Master Mix |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1A | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | A1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 13 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1B | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | B1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 13 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM1 |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1C | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | C1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 13 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM1 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | D1 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 13 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM1 |- style="background-color:#E6B8B7;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1A | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | E1 | align="center" align="center" valign="bottom" | 26 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1B | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | F1 | align="center" align="center" valign="bottom" | 26 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM2 |- style="background-color:#E6B8B7;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1C | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | G1 | align="center" align="center" valign="bottom" | 26 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM2 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | H1 | align="center" align="center" valign="bottom" | 26 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM2 |- style="background-color:#D8E4BC;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1A | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | A12 | align="center" align="center" valign="bottom" | 27 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM3 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1B | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | B12 | align="center" align="center" valign="bottom" | 27 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM3 |- style="background-color:#D8E4BC;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1C | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | C12 | align="center" align="center" valign="bottom" | 27 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM3 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | D12 | align="center" align="center" valign="bottom" | 27 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 0.5 | align="center" align="center" valign="bottom" | 22 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM3 |- style="background-color:#CCC0DA;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1A | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | E12 | align="center" align="center" valign="bottom" | 28 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 2* | align="center" valign="bottom" | 2* | align="center" align="center" valign="bottom" | 19 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM4 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1B | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | F12 | align="center" align="center" valign="bottom" | 28 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 2* | align="center" valign="bottom" | 2* | align="center" align="center" valign="bottom" | 19 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM4 |- style="background-color:#CCC0DA;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1C | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | G12 | align="center" align="center" valign="bottom" | 28 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 2* | align="center" valign="bottom" | 2* | align="center" align="center" valign="bottom" | 19 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM4 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | H12 | align="center" align="center" valign="bottom" | 28 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 2* | align="center" valign="bottom" | 2* | align="center" align="center" valign="bottom" | 19 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM4 |- style="background-color:#FCD5B4;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1A | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | A2 | align="center" align="center" valign="bottom" | 28 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 0.2* | align="center" valign="bottom" | 0.2* | align="center" align="center" valign="bottom" | 22.6 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM5 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1B | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | B2 | align="center" align="center" valign="bottom" | 28 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 0.2* | align="center" valign="bottom" | 0.2* | align="center" align="center" valign="bottom" | 22.6 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM5 |- style="background-color:#FCD5B4;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1C | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | C2 | align="center" align="center" valign="bottom" | 28 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 0.2* | align="center" valign="bottom" | 0.2* | align="center" align="center" valign="bottom" | 22.6 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM5 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | D2 | align="center" align="center" valign="bottom" | 28 | align="center" align="center" valign="bottom" | 0 | align="center" align="center" valign="bottom" | 25 | align="center" valign="bottom" | 0.2* | align="center" valign="bottom" | 0.2* | align="center" align="center" valign="bottom" | 22.6 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MM5 |} <li>Aliquot 48 uL MM1 into each well</li> <li>Add 2 uL sample to appropriate well</li> <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x26</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 184 uL TBE, 46 uL 6x loading dye (2 uL dye/sample and 8 uL TBE/sample, add one sample's worth)</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop<li> <li>Load 10 uL in to well</li> <li>Run gel for 22 minutes at 250V<li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20161003-qPCR-PrimerDilutions.png|qPCR Curve File:2016-10-03-PCAmp4-PrimerDilution1.png|Gel Image 1 File:2016-10-03-PCAmp4-PrimerDilution2.png|Gel Image 2 </gallery> [[Category:ComboLock]] [[Category:20160930]]
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