Daniel:Notebook/ComboLock/2016-8-19: Difference between revisions
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>Djacobse (Created page with "=Positive Control Amplicon Test (Started yesterday)= Back to Calendar ==Protocol== <ol start="2"> <li>...") |
>Djacobse |
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(8 intermediate revisions by the same user not shown) | |||
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<li>Incubate reaction at 37 ºC for 2 hours</li> | <li>Incubate reaction at 37 ºC for 2 hours</li> | ||
<li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> | <li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li> | ||
</ol> | </ol> | ||
<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Set up reaction according to table</li> | <li>Set up reaction according to table</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center" | |||
| width="105" height="45" | Sample | |||
| width="165" | Name | |||
| 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) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 1 | |||
| valign="bottom" | Amp1 Test | |||
| align="center" valign="bottom" | A1 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| 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" | |||
| height="15" valign="bottom" | Sample 2 | |||
| valign="bottom" | Amp3 Test | |||
| align="center" valign="bottom" | B1 | |||
| align="center" align="center" valign="bottom" | 11 | |||
| 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" | |||
| height="15" valign="bottom" | Sample 3 | |||
| valign="bottom" | Latch X2 Specificity | |||
| align="center" valign="bottom" | C1 | |||
| align="center" align="center" valign="bottom" | 12 | |||
| 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" | |||
| height="15" valign="bottom" | Sample 4 | |||
| valign="bottom" | Latch X3 Specificity | |||
| align="center" valign="bottom" | D1 | |||
| align="center" align="center" valign="bottom" | 13 | |||
| 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" | |||
| height="15" valign="bottom" | Sample 5 | |||
| valign="bottom" | No Template | |||
| align="center" valign="bottom" | E1 | |||
| align="center" align="center" valign="bottom" | 14 | |||
| 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" | |||
| height="15" valign="bottom" | Sample 6 | |||
| valign="bottom" | No Latch | |||
| align="center" valign="bottom" | F1 | |||
| align="center" align="center" valign="bottom" | 15 | |||
| 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" | |||
| height="15" valign="bottom" | Sample 7 | |||
| valign="bottom" | No Padlock | |||
| align="center" valign="bottom" | G1 | |||
| align="center" align="center" valign="bottom" | 16 | |||
| 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" | |||
| height="15" valign="bottom" | Sample 8 | |||
| valign="bottom" | PCAmp2 Only | |||
| align="center" valign="bottom" | H1 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| 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" | |||
| height="15" valign="bottom" | Sample 9 | |||
| valign="bottom" | PCR NTC | |||
| align="center" valign="bottom" | A12 | |||
| align="center" align="center" valign="bottom" | 18 | |||
| 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>Master Mix</li> | <li>Master Mix</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> | <li>227.5 uL KAPA SyberFast MM</li> | ||
<li> | <li>159.25 uL nfH2O</li> | ||
<li> | <li>4.55 uL AmpF 6.4</li></ol> | ||
<li>Add 43 uL Master Mix to each well</li> | <li>Add 43 uL Master Mix to each well</li> | ||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
Line 45: | Line 160: | ||
</ol> | </ol> | ||
</ol> | </ol> | ||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:20160819-qPCR-PosControlTest.png|qPCR Curve | |||
File:2016-08-19-PosControl-qPCR.png|Gel Image | |||
</gallery> | |||
It looks like lane 3 and 4 have bands at the correct expected size (231bp). None of the negative controls (5-8) have it, and lane 1 might also have it. This would indicate lane 2 had an error in the process. I can size select the bands and get ready for library prep if that is the case. | |||
[[Category:ComboLock]] [[Category:20160818]] |
Latest revision as of 20:25, 19 August 2016
Positive Control Amplicon Test (Started yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Set up reaction according to table
- Master Mix
- 227.5 uL KAPA SyberFast MM
- 159.25 uL nfH2O
- 4.55 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- 20160819-qPCR-PosControlTest.png
qPCR Curve
- 2016-08-19-PosControl-qPCR.png
Gel Image
Sample | Name | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | Amp1 Test | A1 | 10 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | Amp3 Test | B1 | 11 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | Latch X2 Specificity | C1 | 12 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | Latch X3 Specificity | D1 | 13 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | No Template | E1 | 14 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | No Latch | F1 | 15 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | No Padlock | G1 | 16 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8 | PCAmp2 Only | H1 | 17 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9 | PCR NTC | A12 | 18 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
Results[edit]
It looks like lane 3 and 4 have bands at the correct expected size (231bp). None of the negative controls (5-8) have it, and lane 1 might also have it. This would indicate lane 2 had an error in the process. I can size select the bands and get ready for library prep if that is the case.