Daniel:Notebook/PosSequencing/2017-6-5: Difference between revisions
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>Djacobse (Created page with "=Aqueous PCR Test (Started Friday June 2)= Back to Calendar Given the results from Daniel:Note...") |
>Djacobse m (→Results) |
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(4 intermediate revisions by the same user not shown) | |||
Line 9: | Line 9: | ||
<ol start="4"> | <ol start="4"> | ||
<li>Baking</li> | <li>Baking</li> | ||
<ol type="A"> | |||
<li>Incubate fraction "E" for 15 minutes at 95C</li> | <li>Incubate fraction "E" for 15 minutes at 95C</li> | ||
<li>Chill on ice prior to adding to qPCR lanes</li> | <li>Chill on ice prior to adding to qPCR lanes</li> | ||
</ol> | |||
<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Make the following | <li>Make the following 6.2X master mix (1X)</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> | <li> 6.2 uL 10 uM BiotinUracilPrimer1 (1)</li> | ||
<li> | <li> 6.2 uL 10 uM Primer2 (1)</li> | ||
<li> | <li> 124 uL 2X Kap SYBR Master Mix (20)</li> | ||
<li> | <li> 99.2 uL nfH<sub>2</sub>O (16)</li></ol> | ||
<li>Aliquot 38 uL master mix to appropriate lanes</li> | <li>Aliquot 38 uL master mix to appropriate lanes</li> | ||
<li>Add 2 uL sample according to plate layout below</li> | <li>Add 2 uL sample according to plate layout below</li> | ||
Line 36: | Line 38: | ||
</ol></ol> | </ol></ol> | ||
</ol> | </ol> | ||
==Results== | |||
<gallery perrow=2 heights=225px widths=315px mode=packed-hover> | |||
File:PlateLayout-20170605-aqPCR-EFraction.png|Plate layout | |||
File:20170605-aqPCR-EFraction-plateCTs.png|Plate CT values | |||
File:20170605-aqPCR-EFraction-plateCTs-normed.png|Plate CT values-normalized | |||
File:20170605-aqPCR-EFraction-rawcurves.png|Raw curves | |||
</gallery> | |||
[[Category:PosSeq]] [[Category:20170602]] |
Latest revision as of 18:29, 9 June 2017
Aqueous PCR Test (Started Friday June 2)[edit]
Given the results from Saturday I'm going to repeat qPCR for the E fraction after baking for 15 minutes.
Protocol[edit]
- Baking
- Incubate fraction "E" for 15 minutes at 95C
- Chill on ice prior to adding to qPCR lanes
- qPCR
- Make the following 6.2X master mix (1X)
- 6.2 uL 10 uM BiotinUracilPrimer1 (1)
- 6.2 uL 10 uM Primer2 (1)
- 124 uL 2X Kap SYBR Master Mix (20)
- 99.2 uL nfH2O (16)
- Aliquot 38 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-20170605-aqPCR-EFraction.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x120
- 72C 2 min
- 16C hold
Results[edit]
- PlateLayout-20170605-aqPCR-EFraction.png
Plate layout
- 20170605-aqPCR-EFraction-plateCTs.png
Plate CT values
- 20170605-aqPCR-EFraction-plateCTs-normed.png
Plate CT values-normalized
- 20170605-aqPCR-EFraction-rawcurves.png
Raw curves