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=AmpR Test= [[Daniel:Notebook/ComboLock|Back to Calendar]] [[Daniel:Notebook/ComboLock/2016-9-29|Yesterday]] I tested the AmpR primer (the names are actually confusing, the name is the primer I'm NOT using. So yesterday I amplified off the circularized product using AmpR (which binds to the LinkerL region of the circularized product) and primer12RC, which binds to the amplified product. Now I'm going to test the amplification of using the AmpF primer. If AmpF is the culprit (which is what I expect) then I should see off-target binding. If the off-target products require both AmpF and AmpR, I shouldn't see off-target product. ==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 AmpF6.4Sol | width="65" | 100 uM primer2 | 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+10 | align="center" valign="bottom" | A1 | align="center" valign="bottom" | NA | 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 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 0B | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | A2 | align="center" valign="bottom" | NA | 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 |- 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+10 | align="center" valign="bottom" | A3 | align="center" valign="bottom" | NA | 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 |- 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" valign="bottom" | NA | 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" | 24 | align="center" align="center" valign="bottom" | 50 |} <li>Make the following master mix</li> <ol type="a"> <li>90.2 uL nfH2O</li> <li>2.05 uL Primer2</li> <li>2.05 uL AmpF6.4Sol</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 heights=300px widths=300px> File:20160930-qPCRCurve-Primer2.png|qPCR curve File:2016-09-30-qPCR-Primer2.png|Gel image </gallery> ===Discussion=== Well, this pretty much confirms it. AmpF is the problem and is producing off-target products. Note that the expected product is the 168 bp band, but a primer dimer caused by the off-target binding would produce a product of approximately 75bp. We see a clear band at 75 bp, meaning that the AmpF-primer2 binding is likely too strong. Hopefully with changing primer2 to primer24 (see [[Daniel:Notebook/ComboLock/2016-9-28|PCAmp4 production]]) for further details.
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