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=PCR Testing (Started [[Daniel:Notebook/ComboLock/2016-9-30|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol start="2"> <li>Circularization</li> <ol type="A"> <li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li></ol> <li>Exonuclease Digestion</li> <ol type="A"> <li>Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio</li> <li>Add 2 μL of exonuclease I/III mix to reaction</li> <li>Mix the reaction by swirling pipette around the well 5 times</li> <li>Incubate reaction at 37 ºC for 2 hours</li> <li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> </ol> <li>qPCR</li> <ol type="A"> <li>Set up reaction according to table in next section</li> <li>Make master mixes according to following recipes</li> <ol type="a"> <li>334.25 uL nfH2O</li> <li>477.5 uL SYBR Fast</li> <li>9.55 uL AmpF</li> </ol> <li>Add 43 uL master mix 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 x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol></ol> ===qPCR Sample Table=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#948A54;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 AmpF 6.4 | 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+10 | align="center" valign="bottom" | A1 | align="center" align="center" valign="bottom" | 24 | 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+10 | align="center" valign="bottom" | A2 | align="center" align="center" valign="bottom" | 24 | 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+10 | align="center" valign="bottom" | A3 | align="center" align="center" valign="bottom" | 24 | 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 1A | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | A4 | 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" | 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 1B | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | A5 | 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" | 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 1C | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | A6 | 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" | 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:#B8CCE4;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2A | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | A7 | 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" | 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 2B | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | A8 | 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" | 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:#B8CCE4;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2C | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | H1 | 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" | 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 3A | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | H2 | 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" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#B8CCE4;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 3B | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | H3 | 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" | 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 3C | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | H4 | 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" | 5 | align="center" align="center" valign="bottom" | 17.5 | align="center" align="center" valign="bottom" | 50 |- style="background-color:#E6B8B7;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4A | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | H5 | align="center" align="center" valign="bottom" | 28 | 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 4B | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | H6 | align="center" align="center" valign="bottom" | 28 | 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:#E6B8B7;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 4C | align="center" align="center" valign="bottom" | 1.00E+10 | align="center" valign="bottom" | H7 | align="center" align="center" valign="bottom" | 28 | 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 5A | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | H8 | align="center" align="center" valign="bottom" | 29 | 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:#E6B8B7;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 5B | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | D1 | align="center" align="center" valign="bottom" | 29 | 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 5C | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | D2 | align="center" align="center" valign="bottom" | 29 | 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:#E6B8B7;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | D3 | align="center" align="center" valign="bottom" | 30 | 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 |} ===Results=== Note: I stopped the qPCR reaction for lanes A1-H1 (Samples 0A-C,1A-C,2A,2B) at cycle 24 since they had reached saturation. <gallery perrow=2 widths=300px heights=300px> File:20161001-qPCR-LatchX4Test1.png|qPCR Curve File:|No file File:2016-10-01-PCAmpX4Test.png|Gel Image 1 File:2016-10-01-PCAmpX4Test2.png|Gel Image 2 </gallery> ===Discussion=== Well, this is a mixed success. The 100 amol sample (1E7 molecules) has 231bp product in all three lanes, and the 10 amol samples (1E6) have signal in 1, kinda 2 lanes. This is a 100-1000 fold signal increase, which is great. There are however, still prevalent primer dimers at ~100bp that are present even in the NTC lane. Addressing these primer dimers will probably increase signal to an even higher level. As it stands 1E7 is approximately 1000 copies of an mRNA (or protein) in an experiment with 10,000 cells. 1E6 is a 100 copy mRNA/protein. [[Category:ComboLock]] [[Category:20160930]]
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