Daniel:Notebook/ComboLock/2016-6-24: Difference between revisions
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>Djacobse |
>Djacobse |
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<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="134" height="45" | Sample | |||
| width="65" | Lane | |||
| width="65" | Batch | |||
| width="65" | Cells | |||
| width="65" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 100 uM Primer2 | |||
| width="65" | 100 uM Primer4-RC | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 1 | |||
| align="center" valign="bottom" | A1 | |||
| align="center" valign="bottom" | B1 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 22 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 2 | |||
| align="center" valign="bottom" | A2 | |||
| align="center" valign="bottom" | B2 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 22 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 3 | |||
| align="center" valign="bottom" | A3 | |||
| align="center" valign="bottom" | B3 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 22 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 4 (No Probe) | |||
| align="center" valign="bottom" | A4 | |||
| align="center" valign="bottom" | B4 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 22 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|} | |||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
<ol type="a"> | <ol type="a"> |
Revision as of 23:54, 22 June 2016
Stage 2 Test (Started yesterday)
Protocol
- Latch and Padlock Hybridization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 2 uL exonuclease I (20 units/μL) and 10 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
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
Sample | Lane | Batch | Cells | 2X Kapa SYBR qPCR MM | 100 uM Primer2 | 100 uM Primer4-RC | H2O | Total Volume (uL) |
Sample 1 | A1 | B1 | 1 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 2 | A2 | B2 | 1 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 3 | A3 | B3 | 1 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 4 (No Probe) | A4 | B4 | 1 | 25 | 0.5 | 0.5 | 22 | 50 |