Daniel:Notebook/ComboLock/2016-10-4: Difference between revisions
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>Djacobse (Created page with "=Primer24 qPCR Test (From Yesterday)= Back to Calendar Continuing the primer amplifications to test off...") |
>Djacobse |
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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| 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" | |- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="105" height="45" | Sample | ||
| width="65" | Molecule Count | | width="65" | Molecule Count | ||
| width="65" | Lane | | width="65" | Lane | ||
Line 72: | Line 72: | ||
<li>Make the following master mix</li> | <li>Make the following master mix</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> | <li>90.2 uL nfH2O</li> | ||
<li>2.05 uL 100 uM primer12 RC</li> | <li>2.05 uL 100 uM primer12 RC</li> | ||
<li> | <li>2.05 uL 10 uM Amp6.3R Index 30</li> | ||
<li>102.5 uL Kapa SYBR Fast</li></ol> | <li>102.5 uL Kapa SYBR Fast</li></ol> | ||
<li>Aliquot 43 uL MM1 into each well</li> | <li>Aliquot 43 uL MM1 into each well</li> | ||
Line 93: | Line 93: | ||
<li>Mix 48 uL TBE, 12 uL 6x loading dye</li> | <li>Mix 48 uL TBE, 12 uL 6x loading dye</li> | ||
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | ||
<li>Add 2 uL of sample or ladder to correct drop<li> | <li>Add 2 uL of sample or ladder to correct drop</li> | ||
<li>Load 10 uL in to well</li> | <li>Load 10 uL in to well</li> | ||
<li>Run gel for 22 minutes at 250V<li> | <li>Run gel for 22 minutes at 250V</li> | ||
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | ||
<li>Rinse gel and image in gel doc</li> | <li>Rinse gel and image in gel doc</li> | ||
</ol></ol> | </ol></ol> | ||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:2016-10-04-qPCR-LatchX4-Primer24-AmpF.png|qPCR Curve | |||
File:2016-10-04-LatchX4-AmpF-Primer24.png|Gel image | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20160930]] |
Latest revision as of 18:41, 7 October 2016
Primer24 qPCR Test (From Yesterday)[edit]
Continuing the primer amplifications to test off-target binding.
Protocol[edit]
- qPCR
- Set up qPCR reactions according to table
- Make the following master mix
- 90.2 uL nfH2O
- 2.05 uL 100 uM primer12 RC
- 2.05 uL 10 uM Amp6.3R Index 30
- 102.5 uL Kapa SYBR Fast
- Aliquot 43 uL MM1 into each well
- Add 2 uL sample to appropriate well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 48 uL TBE, 12 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Molecule Count | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM primer24 | 100 uM AmpF | H2O | Total Volume (uL) |
Sample 0A | 1.00E+09 | A1 | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 0B | 1.00E+09 | A2 | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 0C | 1.00E+09 | A3 | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
NTC (rev) | 0 | A4 | 0 | 25 | 0.5 | 0.5 | 22 | 50 |
Results[edit]
- 2016-10-04-qPCR-LatchX4-Primer24-AmpF.png
qPCR Curve
- 2016-10-04-LatchX4-AmpF-Primer24.png
Gel image