Daniel:Notebook/ComboLock/2016-9-26: Difference between revisions
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[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
Continuing the tests on qPCR for the positive control amplicon using primer concentrations. This is following some of the conclusions from [[Daniel:Notebook/ComboLock/ | Continuing the tests on qPCR for the positive control amplicon using primer concentrations. This is following some of the conclusions from [[Daniel:Notebook/ComboLock/2016-9-23|Friday]]. | ||
==Protocol== | ==Protocol== | ||
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| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 21 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | NTC | | align="center" valign="bottom" | NTC | ||
Line 178: | Line 178: | ||
| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 22 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | NTC | | align="center" valign="bottom" | NTC | ||
Line 189: | Line 189: | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| | | align="center" valign="bottom" | 2* | ||
| | | align="center" valign="bottom" | 2* | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 19 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | MM4 | | align="center" valign="bottom" | MM4 | ||
Line 202: | Line 202: | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| | | align="center" valign="bottom" | 2* | ||
| | | align="center" valign="bottom" | 2* | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 19 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | MM4 | | align="center" valign="bottom" | MM4 | ||
Line 215: | Line 215: | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| | | align="center" valign="bottom" | 2* | ||
| | | align="center" valign="bottom" | 2* | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 19 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | MM4 | | align="center" valign="bottom" | MM4 | ||
Line 228: | Line 228: | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| | | align="center" valign="bottom" | 2* | ||
| | | align="center" valign="bottom" | 2* | ||
| align="center" align="center" valign="bottom" | 19 | | align="center" align="center" valign="bottom" | 19 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | NTC | | align="center" valign="bottom" | NTC | ||
Line 241: | Line 241: | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| | | align="center" valign="bottom" | 0.2* | ||
| | | align="center" valign="bottom" | 0.2* | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 22.6 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | MM5 | | align="center" valign="bottom" | MM5 | ||
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| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| | | align="center" valign="bottom" | 0.2* | ||
| | | align="center" valign="bottom" | 0.2* | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 22.6 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | MM5 | | align="center" valign="bottom" | MM5 | ||
Line 267: | Line 267: | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| | | align="center" valign="bottom" | 0.2* | ||
| | | align="center" valign="bottom" | 0.2* | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 22.6 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | MM5 | | align="center" valign="bottom" | MM5 | ||
Line 280: | Line 280: | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
| align="center" align="center" valign="bottom" | 25 | | align="center" align="center" valign="bottom" | 25 | ||
| | | align="center" valign="bottom" | 0.2* | ||
| | | align="center" valign="bottom" | 0.2* | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 22.6 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
| align="center" valign="bottom" | NTC | | align="center" valign="bottom" | NTC | ||
Line 289: | Line 289: | ||
<li>Make master mixes according to following table</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="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" valign="bottom" | |||
| width="140" rowspan="2" height="15" | Reagent | |||
| width="65" colspan="5" | Master Mix | |||
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" valign="bottom" | |||
| MM1 | |||
| MM2 | |||
| MM3 | |||
| MM4 | |||
| MM5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | PrimerF (10 uM) | |||
| align="center" align="center" valign="bottom" | 15.5 | |||
| align="center" align="center" valign="bottom" | 3.1 | |||
| align="center" align="center" valign="bottom" | 1.55 | |||
| align="center" valign="bottom" | 6.2* | |||
| align="center" valign="bottom" | 0.62* | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | PrimerR (10 uM) | |||
| align="center" align="center" valign="bottom" | 15.5 | |||
| align="center" align="center" valign="bottom" | 3.1 | |||
| align="center" align="center" valign="bottom" | 1.55 | |||
| align="center" valign="bottom" | 6.2* | |||
| align="center" valign="bottom" | 0.62* | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | KAPA Master Mix | |||
| align="center" align="center" valign="bottom" | 77.5 | |||
| align="center" align="center" valign="bottom" | 77.5 | |||
| align="center" align="center" valign="bottom" | 77.5 | |||
| align="center" align="center" valign="bottom" | 77.5 | |||
| align="center" align="center" valign="bottom" | 77.5 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 40.3 | |||
| align="center" align="center" valign="bottom" | 65.1 | |||
| align="center" align="center" valign="bottom" | 68.2 | |||
| align="center" align="center" valign="bottom" | 58.9 | |||
| align="center" align="center" valign="bottom" | 70.06 | |||
|} | |||
<li>Add 48 uL correct master mix to each well</li> | <li>Add 48 uL correct master mix to each well</li> | ||
Line 295: | Line 341: | ||
<li>95C 3 min</li> | <li>95C 3 min</li> | ||
<li>95C 3 sec</li> | <li>95C 3 sec</li> | ||
<li> | <li>60C 30 sec</li> | ||
<li>72C 20 sec</li> | <li>72C 20 sec</li> | ||
<li>plate read</li> | <li>plate read</li> | ||
Line 302: | Line 348: | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol></ol> | </ol></ol> | ||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:2016-09-26-PosConPrimersA.png|First Gel | |||
File:2016-09-26-PosConPrimersB.png|Second Gel | |||
File:20160926qPCR_PrimerConc.png|qPCR Curve; averaged across 3 lanes for samples | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20160922]] | [[Category:ComboLock]] [[Category:20160922]] |
Latest revision as of 23:13, 29 September 2016
PCR Testing (Started Thursday)[edit]
Continuing the tests on qPCR for the positive control amplicon using primer concentrations. This is following some of the conclusions from Friday.
Protocol[edit]
- qPCR
- Follow reaction map below
- Make master mixes according to following table
- Add 48 uL correct master mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 60C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
- 2016-09-26-PosConPrimersA.png
First Gel
- 2016-09-26-PosConPrimersB.png
Second Gel
- 20160926qPCR PrimerConc.png
qPCR Curve; averaged across 3 lanes for samples
Sample | Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) | Master Mix |
Sample 1A | 1.00E+09 | A1 | 36 | 2 | 25 | 5 | 5 | 13 | 50 | MM1 |
Sample 1B | 1.00E+09 | A2 | 36 | 2 | 25 | 5 | 5 | 13 | 50 | MM1 |
Sample 1C | 1.00E+09 | A3 | 36 | 2 | 25 | 5 | 5 | 13 | 50 | MM1 |
NTC | 0 | A4 | 36 | 0 | 25 | 5 | 5 | 13 | 50 | NTC |
Sample 1A | 1.00E+09 | A5 | 36 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1B | 1.00E+09 | A6 | 36 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1C | 1.00E+09 | A7 | 36 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
NTC | 0 | A8 | 36 | 0 | 25 | 1 | 1 | 21 | 50 | NTC |
Sample 1A | 1.00E+09 | H1 | 36 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 1B | 1.00E+09 | H2 | 36 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 1C | 1.00E+09 | H3 | 36 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
NTC | 0 | H4 | 36 | 0 | 25 | 0.5 | 0.5 | 22 | 50 | NTC |
Sample 1A | 1.00E+09 | H5 | 37 | 2 | 25 | 2* | 2* | 19 | 50 | MM4 |
Sample 1B | 1.00E+09 | H6 | 37 | 2 | 25 | 2* | 2* | 19 | 50 | MM4 |
Sample 1C | 1.00E+09 | H7 | 37 | 2 | 25 | 2* | 2* | 19 | 50 | MM4 |
NTC | 0 | H8 | 37 | 0 | 25 | 2* | 2* | 19 | 50 | NTC |
Sample 1A | 1.00E+09 | C1 | 37 | 2 | 25 | 0.2* | 0.2* | 22.6 | 50 | MM5 |
Sample 1B | 1.00E+09 | C2 | 37 | 2 | 25 | 0.2* | 0.2* | 22.6 | 50 | MM5 |
Sample 1C | 1.00E+09 | C3 | 37 | 2 | 25 | 0.2* | 0.2* | 22.6 | 50 | MM5 |
NTC | 0 | C4 | 37 | 0 | 25 | 0.2* | 0.2* | 22.6 | 50 | NTC |
Reagent | Master Mix | ||||
MM1 | MM2 | MM3 | MM4 | MM5 | |
PrimerF (10 uM) | 15.5 | 3.1 | 1.55 | 6.2* | 0.62* |
PrimerR (10 uM) | 15.5 | 3.1 | 1.55 | 6.2* | 0.62* |
KAPA Master Mix | 77.5 | 77.5 | 77.5 | 77.5 | 77.5 |
nfH2O | 40.3 | 65.1 | 68.2 | 58.9 | 70.06 |