Daniel:Notebook/ComboLock/2016-10-1: Difference between revisions
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>Djacobse (Created page with "=PCR Testing (Started Yesterday)= Back to Calendar ==Protocol== <ol start="2"> <li>Circularization</li...") |
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<ol type="A"> | <ol type="A"> | ||
<li>Set up reaction according to table in next section</li> | <li>Set up reaction according to table in next section</li> | ||
<li>Make master mixes according to following | <li>Make master mixes according to following recipes</li> | ||
<ol type="a"> | |||
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===qPCR Sample Table=== | ===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" | 21.9 | |||
| 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" | 21.9 | |||
| 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" | 21.9 | |||
| 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" | B1 | |||
| 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" | B2 | |||
| 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" | B3 | |||
| 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" | B4 | |||
| 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" | 21.9 | |||
| 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" | B5 | |||
| 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" | 21.9 | |||
| 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" | B6 | |||
| 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" | 21.9 | |||
| 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" | D1 | |||
| 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" | D2 | |||
| 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" | D3 | |||
| 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" | D4 | |||
| 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" | 21.9 | |||
| 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" | D5 | |||
| 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" | 21.9 | |||
| 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" | D6 | |||
| 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" | 21.9 | |||
| 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" | D7 | |||
| 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 | |||
|} |
Revision as of 20:40, 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
- Add 48 uL correct 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 | 21.9 | 50 |
Sample 1B | 1.00E+09 | A5 | 25 | 2 | 25 | 0.5 | 5 | 21.9 | 50 |
Sample 1C | 1.00E+09 | A6 | 25 | 2 | 25 | 0.5 | 5 | 21.9 | 50 |
Sample 2A | 1.00E+10 | B1 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2B | 1.00E+10 | B2 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2C | 1.00E+10 | B3 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3A | 1.00E+09 | B4 | 27 | 2 | 25 | 0.5 | 5 | 21.9 | 50 |
Sample 3B | 1.00E+09 | B5 | 27 | 2 | 25 | 0.5 | 5 | 21.9 | 50 |
Sample 3C | 1.00E+09 | B6 | 27 | 2 | 25 | 0.5 | 5 | 21.9 | 50 |
Sample 4A | 1.00E+10 | D1 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4B | 1.00E+10 | D2 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4C | 1.00E+10 | D3 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5A | 1.00E+09 | D4 | 29 | 2 | 25 | 0.5 | 5 | 21.9 | 50 |
Sample 5B | 1.00E+09 | D5 | 29 | 2 | 25 | 0.5 | 5 | 21.9 | 50 |
Sample 5C | 1.00E+09 | D6 | 29 | 2 | 25 | 0.5 | 5 | 21.9 | 50 |
NTC | 0 | D7 | 30 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |