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=Positive Control Amplicon Production (PCAmp4; Started [[Daniel:Notebook/ComboLock/2016-9-28|yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol start="4"> <li>Size Selection</li> <ol type="A" start="13"> <li>Centrifuge at 12000 rpm for 1.5 minutes</li> <li>Extract the supernatant and place into NanoSep column</li> <li>Centrifuge at 12000 rpm for 1.5 minutes</li> </ol> <li>Ethanol Precipitation</li> <ol type="A"> <li>Move sample to a fresh 2 mL tube</li> <li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc and 2 uL glycoblue</li> <li>Store at for 6 hours</li> <li>Pellet by centrifuging for 25 minutes at 12000rpm at 4C</li> <li>Remove supernatant and add 750 uL chilled 70% EtOH</li> <li>Centrifuge for 12 minutes at 12000rpm at 4C</li> <li>Remove supernatant and spin in vacuum centrifuge for 10 minutes</li> <li>Add 50 uL nfH2O and resuspend every tube in series; <li>Measure final concentration in nanodrop</li> </ol></ol> ===Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4BD97;font-size:12pt;font-weight:bold" align="center" | width="155" height="27" | Sample | width="77" | ng/uL ssDNA |- style="font-size:12pt" | height="15" valign="bottom" | C4-C2 Amplicon (Final) | align="center" align="center" valign="bottom" | 78.3 |} =AmpF Test= Since I'm testing whether the AmpF primer (which has LinkerR that matches to primer2 and therefore has a potential off-target binding) creates the wrong product or not. To test this I'll be using the PCR primer Primer12-RC, which should be a forward primer for amplifying off the circular target. In conjuction with AmpR6.3, this should produce a 157bp product. Let's see what happens. ==Protocol== <ol> <li>qPCR</li> <ol type="A"> <li>Follow reaction table below to set up 4 reactions</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="65" | 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 1A | 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 1B | 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 1C | 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 | 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 Primer12RC</li> <li>20.5 uL AmpR6.3-34</li> <li>102.5 uL SYBR Fast</li></ol> <li>Aliquot 48 uL master mix into each well</li> <li>Add 2 uL sample to each 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> ===Results=== <gallery perrow=2 widths=300px heights=300px> File:20160929-qPCRAmpFTest.png|qPCR Curve File:2016-09-29-qPCR-Primer12RC.png|Gel Image </gallery> ===Discussion=== As I expected there is no extra product (or very little) being formed. [[Category:ComboLock]] [[Category:20160928]] [[Category:20160929]]
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