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Daniel:Notebook/ComboLock/2016-9-23
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=PCR Testing (Started [[Daniel:Notebook/ComboLock/2016-9-22|Yesterday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol (all)== <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></ol> Standard qPCR like I normally do. Control version for the tests after. <ol start="4"> <li>qPCR</li> <ol type="A"> <li>Follow reaction map below</li> *Note:"X"=5 uL primer per reaction; "O" indicates 1 uL primer *Note:"TN" is a range of temperatures (gradient) from 50C (Lane 1) to 65C (Lane 12) {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" valign="bottom" | align="center" width="65" height="35" | |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 1 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 2 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 3 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 4 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 5 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 6 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 7 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 8 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 9 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 10 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 11 |style="background-color:#FCD5B4;font-weight:bold" align="center" width="65" | 12 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | A | X,T1 | align="center" | | X,T2 | align="center" | | X,T3 | align="center" | | X,T4 | align="center" | | X,T5 | align="center" | | align="center" | | X,T6 |- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | B | X,T1 | align="center" | | X,T2 | align="center" | | X,T3 | align="center" | | X,T4 | align="center" | | X,T5 | align="center" | | align="center" | | X,T6 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | C | X,T1 | align="center" | | X,T2 | align="center" | | X,T3 | align="center" | | X,T4 | align="center" | | X,T5 | align="center" | | align="center" | | X,T6 |- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | D | O,T1 | align="center" | | O,T2 | align="center" | | O,T3 | align="center" | | O,T4 | align="center" | | O,T5 | align="center" | | align="center" | | O,T6 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | E | O,T1 | align="center" | | O,T2 | align="center" | | O,T3 | align="center" | | O,T4 | align="center" | | O,T5 | align="center" | | align="center" | | O,T6 |- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | F | O,T1 | align="center" | | O,T2 | align="center" | | O,T3 | align="center" | | O,T4 | align="center" | | O,T5 | align="center" | | align="center" | | O,T6 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | G | NTC | align="center" | | NTC | align="center" | | NTC | align="center" | | NTC | align="center" | | NTC | align="center" | | align="center" | | NTC |- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | H | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | |} <li>Set up reaction according to table in next section</li> <li>Make master mixes according to following table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" | align="center" width="129" height="22" valign="bottom" | |style="background-color:#B8CCE4" width="65" colspan="3" align="center" valign="bottom" | Master Mix |- style="font-size:12pt" | height="15" valign="bottom" | Reagent |style="background-color:#B8CCE4" align="center" valign="bottom" | MMX |style="background-color:#B8CCE4" align="center" valign="bottom" | MMO |style="background-color:#B8CCE4" align="center" valign="bottom" | NTC |- style="font-size:12pt" | height="15" valign="bottom" | PrimerF (100 uM) | align="center" align="center" valign="bottom" | 9.05 | align="center" align="center" valign="bottom" | 1.81 | align="center" align="center" valign="bottom" | 3.05 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | PrimerR (10 uM) | align="center" align="center" valign="bottom" | 90.5 | align="center" align="center" valign="bottom" | 18.1 | align="center" align="center" valign="bottom" | 30.5 |- style="font-size:12pt" | height="15" valign="bottom" | KAPA Master Mix | align="center" align="center" valign="bottom" | 452.5 | align="center" align="center" valign="bottom" | 452.5 | align="center" align="center" valign="bottom" | 152.5 |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 316.75 | align="center" align="center" valign="bottom" | 396.39 | align="center" align="center" valign="bottom" | 118.95 |} <li>Add 48 uL correct 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> ===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="65" | 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) | 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" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | A2 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | A3 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- 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" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- 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" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | A7 | align="center" align="center" valign="bottom" | 36 | 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 | align="center" valign="bottom" | NTC |- style="background-color:#B8CCE4;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" | B1 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | 34 | 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 | align="center" valign="bottom" | MMX |- style="background-color:#B8CCE4;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" | B3 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | B4 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#B8CCE4;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" | B5 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- 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" | B6 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#B8CCE4;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | B7 | align="center" align="center" valign="bottom" | 36 | 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 | align="center" valign="bottom" | NTC |- 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" | D1 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | D2 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | D3 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | D4 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#E6B8B7;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" | D5 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- 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" | D6 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- 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" | D7 | align="center" align="center" valign="bottom" | 36 | 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 | align="center" valign="bottom" | NTC |- style="background-color:#D8E4BC;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" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | B7 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- style="background-color:#D8E4BC;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" | C7 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2A | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | D7 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#D8E4BC;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | E7 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2C | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | F7 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#D8E4BC;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | H7 | align="center" align="center" valign="bottom" | 36 | 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 | align="center" valign="bottom" | NTC |- style="background-color:#FCD5B4;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" | A9 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | B9 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- style="background-color:#FCD5B4;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" | C9 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2A | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | D9 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#FCD5B4;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | E9 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2C | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | F9 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#FCD5B4;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" align="center" valign="bottom" | 0 | align="center" valign="bottom" | H9 | align="center" align="center" valign="bottom" | 36 | 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 | align="center" valign="bottom" | NTC |- style="background-color:#CCC0DA;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" | A12 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- 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" | B12 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- style="background-color:#CCC0DA;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" | C12 | align="center" align="center" valign="bottom" | 34 | 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 | align="center" valign="bottom" | MMX |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2A | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | D12 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#CCC0DA;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | E12 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2C | align="center" align="center" valign="bottom" | 1.00E+09 | align="center" valign="bottom" | F12 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 25 | align="center" align="center" valign="bottom" | 0.1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 21.9 | align="center" align="center" valign="bottom" | 50 | align="center" valign="bottom" | MMO |- style="background-color:#CCC0DA;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" | 36 | 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 | align="center" valign="bottom" | NTC |} ===Results=== The qPCR lanes are averages for sample lanes (not for NTCs) <gallery perrow=3 heights=300px widths=300px> File:2016-09-23_16-PosCon-65C.png|65C Annealing temp File:2016-09-23_16-PosCon-64C.png|64C Annealing temp File:2016-09-23_16-PosCon-59C.png|59C Annealing temp File:2016-09-23_16-PosCon-55C.png|55C Annealing temp File:2016-09-23_16-PosCon-51C.png|51C Annealing temp File:2016-09-23_16-PosCon-50C.png|50C Annealing temp File:20160923qPCR_SamplevsNTC.png|Samples (blue) vs NTC; different marker symbols indicate different temperatures File:20160923qPCR_PrimerConc.png|Primer Concentration effects; 1 uM primers in light blue and 0.2 uM primer samples in dark blue File:20160923qPCR_TempHiPrimer.png|Temperature dependence for 1 uM primer set File:20160923qPCR_TempLoPrimer.png|Temperature dependence for 0.2 uM primer set </gallery> ===Discussion=== Well, I'm not sure there is a clear indication, at least at the temperature range I used, that there is a temperature dependence. Comparing the 65C (theoretically the most stringent) and the 50C (least stringent) show little difference overall between the off-target bands. The lowered primer concentration does seem to help though, so I can try again on Monday with a continually decreasing level of primers. This also raises a point that the NTC consistently showed primer-dimer signal (~110bp). Looking back at some of the previous gels with NTCs ([[Daniel:Notebook/ComboLock/2016-8-19|this]] one shows it best), I think that the primer-dimers do form on their own but are helped by the presence of the padlock especially. Of note however is also that the number of cycles for this test was higher than usual. From previous PCR curves (see example from the [[Daniel:Notebook/ComboLock/2016-8-30|X3 dilution test]]) you can see that I went an extra 6 cycles or so and that the NTC lanes start to amplify around cycle 23. [[Category:ComboLock]] [[Category:20160922]]
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