Daniel:Notebook/ComboLock/2016-10-1: Difference between revisions
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<li>Make master mixes according to following recipes</li> | <li>Make master mixes according to following recipes</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li>334.25 uL nfH2O</li> | |||
<li>477.5 uL SYBR Fast</li> | |||
<li>9.55 uL AmpF</li> | |||
<li>Add | </ol> | ||
<li>Add 43 uL master mix to each well</li> | |||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
<ol type="a"> | <ol type="a"> | ||
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<li>72C 2 min</li> | <li>72C 2 min</li> | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol></ol> | </ol></ol></ol> | ||
===qPCR Sample Table=== | ===qPCR Sample Table=== | ||
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| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
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| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
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| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 2A | |style="font-weight:bold" height="15" valign="bottom" | Sample 2A | ||
| align="center" align="center" valign="bottom" | 1.00E+10 | | align="center" align="center" valign="bottom" | 1.00E+10 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | A7 | ||
| align="center" align="center" valign="bottom" | 26 | | align="center" align="center" valign="bottom" | 26 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
Line 140: | Line 141: | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2B | |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | ||
| align="center" align="center" valign="bottom" | 1.00E+10 | | align="center" align="center" valign="bottom" | 1.00E+10 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | A8 | ||
| align="center" align="center" valign="bottom" | 26 | | align="center" align="center" valign="bottom" | 26 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 2C | |style="font-weight:bold" height="15" valign="bottom" | Sample 2C | ||
| align="center" align="center" valign="bottom" | 1.00E+10 | | align="center" align="center" valign="bottom" | 1.00E+10 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | H1 | ||
| align="center" align="center" valign="bottom" | 26 | | align="center" align="center" valign="bottom" | 26 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 3A | |style="font-weight:bold" height="15" valign="bottom" | Sample 3A | ||
| align="center" align="center" valign="bottom" | 1.00E+09 | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | H2 | ||
| align="center" align="center" valign="bottom" | 27 | | align="center" align="center" valign="bottom" | 27 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 3B | |style="font-weight:bold" height="15" valign="bottom" | Sample 3B | ||
| align="center" align="center" valign="bottom" | 1.00E+09 | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | H3 | ||
| align="center" align="center" valign="bottom" | 27 | | align="center" align="center" valign="bottom" | 27 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 3C | |style="font-weight:bold" height="15" valign="bottom" | Sample 3C | ||
| align="center" align="center" valign="bottom" | 1.00E+09 | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | H4 | ||
| align="center" align="center" valign="bottom" | 27 | | align="center" align="center" valign="bottom" | 27 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 4A | |style="font-weight:bold" height="15" valign="bottom" | Sample 4A | ||
| align="center" align="center" valign="bottom" | 1.00E+10 | | align="center" align="center" valign="bottom" | 1.00E+10 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | H5 | ||
| align="center" align="center" valign="bottom" | 28 | | align="center" align="center" valign="bottom" | 28 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 4B | |style="font-weight:bold" height="15" valign="bottom" | Sample 4B | ||
| align="center" align="center" valign="bottom" | 1.00E+10 | | align="center" align="center" valign="bottom" | 1.00E+10 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | H6 | ||
| align="center" align="center" valign="bottom" | 28 | | align="center" align="center" valign="bottom" | 28 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 4C | |style="font-weight:bold" height="15" valign="bottom" | Sample 4C | ||
| align="center" align="center" valign="bottom" | 1.00E+10 | | align="center" align="center" valign="bottom" | 1.00E+10 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | H7 | ||
| align="center" align="center" valign="bottom" | 28 | | align="center" align="center" valign="bottom" | 28 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 5A | |style="font-weight:bold" height="15" valign="bottom" | Sample 5A | ||
| align="center" align="center" valign="bottom" | 1.00E+09 | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | H8 | ||
| align="center" align="center" valign="bottom" | 29 | | align="center" align="center" valign="bottom" | 29 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
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|style="font-weight:bold" height="15" valign="bottom" | Sample 5B | |style="font-weight:bold" height="15" valign="bottom" | Sample 5B | ||
| align="center" align="center" valign="bottom" | 1.00E+09 | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | D1 | ||
| align="center" align="center" valign="bottom" | 29 | | align="center" align="center" valign="bottom" | 29 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
Line 260: | Line 261: | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 5C | |style="font-weight:bold" height="15" valign="bottom" | Sample 5C | ||
| align="center" align="center" valign="bottom" | 1.00E+09 | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | D2 | ||
| align="center" align="center" valign="bottom" | 29 | | align="center" align="center" valign="bottom" | 29 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
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| align="center" align="center" valign="bottom" | 0.5 | | align="center" align="center" valign="bottom" | 0.5 | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 17.5 | ||
| align="center" align="center" valign="bottom" | 50 | | align="center" align="center" valign="bottom" | 50 | ||
Line 272: | Line 273: | ||
|style="font-weight:bold" height="15" valign="bottom" | NTC | |style="font-weight:bold" height="15" valign="bottom" | NTC | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
| align="center" valign="bottom" | | | align="center" valign="bottom" | D3 | ||
| align="center" align="center" valign="bottom" | 30 | | align="center" align="center" valign="bottom" | 30 | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 |
Revision as of 20:44, 1 October 2016
PCR Testing (Started Yesterday)
Protocol
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- 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
- 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 in next section
- Make master mixes according to following recipes
- 334.25 uL nfH2O
- 477.5 uL SYBR Fast
- 9.55 uL AmpF
- 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 x24
- 72C 2 min
- 16C hold
qPCR Sample Table
Sample | Molecule Count | 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 0A | 1.00E+10 | A1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 0B | 1.00E+10 | A2 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 0C | 1.00E+10 | A3 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1A | 1.00E+09 | A4 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | 1.00E+09 | A5 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1C | 1.00E+09 | A6 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2A | 1.00E+10 | A7 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2B | 1.00E+10 | A8 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2C | 1.00E+10 | H1 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3A | 1.00E+09 | H2 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3B | 1.00E+09 | H3 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3C | 1.00E+09 | H4 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4A | 1.00E+10 | H5 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4B | 1.00E+10 | H6 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4C | 1.00E+10 | H7 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5A | 1.00E+09 | H8 | 29 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5B | 1.00E+09 | D1 | 29 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5C | 1.00E+09 | D2 | 29 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | D3 | 30 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |