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
 
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<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 14.2X master mix (1X)</li>
<li>Make the following 6.2X master mix (1X)</li>
<ol type="a">
<ol type="a">
<li> 14.2 uL 10 uM BiotinUracilPrimer1 (1)</li>
<li> 6.2 uL 10 uM BiotinUracilPrimer1 (1)</li>
<li> 14.2 uL 10 uM Primer2 (1)</li>
<li> 6.2 uL 10 uM Primer2 (1)</li>
<li> 284 uL 2X Kap SYBR Master Mix (20)</li>
<li> 124 uL 2X Kap SYBR Master Mix (20)</li>
<li> 227.2 uL nfH<sub>2</sub>O (16)</li></ol>
<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>
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</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]

Back to Calendar

Given the results from Saturday I'm going to repeat qPCR for the E fraction after baking for 15 minutes.

Protocol[edit]

  1. Baking
    1. Incubate fraction "E" for 15 minutes at 95C
    2. Chill on ice prior to adding to qPCR lanes
  2. qPCR
    1. Make the following 6.2X master mix (1X)
      1. 6.2 uL 10 uM BiotinUracilPrimer1 (1)
      2. 6.2 uL 10 uM Primer2 (1)
      3. 124 uL 2X Kap SYBR Master Mix (20)
      4. 99.2 uL nfH2O (16)
    2. Aliquot 38 uL master mix to appropriate lanes
    3. Add 2 uL sample according to plate layout below
    4. File:PlateLayout-20170605-aqPCR-EFraction.png
    5. Run the following thermocycler program
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x120
      7. 72C 2 min
      8. 16C hold

Results[edit]