Daniel:Notebook/ComboLock/2017-5-1: Difference between revisions

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>Djacobse
(Created page with "=Production Run (Started Thursday 4/27)= Back to Calendar <gallery perrow=2 heights=300px widths=300px>...")
 
>Djacobse
 
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=Production Run (Started [[Daniel:Notebook/ComboLock/2017-4-29|Thursday 4/27]])=
=Production Run (Started [[Daniel:Notebook/ComboLock/2017-4-28|Friday 4/28]])=


[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]


<gallery perrow=2 heights=300px widths=300px>
==Protocol-Part 5==
 
<ol start="8">
<li>qPCR</li>
<ol type="A">
<li>Make 17.2X master mix according to following (1X)</li>
<ol type="a">
<li>361.2 uL nfH<sub>2</sub>O (21)</li>
<li>17.2 uL 10 uM AmpF6.4Sol (1)</li>
<li>430 uL 2X Kapa SYBR Fast Master Mix (25)</li></ol>
<li>Add 47 uL master mix to each well</li>
<li>Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout</li>
 
[[Image:PlateLayout-20170501-ProductionRun-PostBglii.png|600px]]
 
<li>qPCR Cycles</li>
<ol type="a">
<li>95C 3 min</li>
<li>95C 3 sec</li>
<li>55C 30 sec</li>
<li>72C 20 sec</li>
<li>plate read</li>
<li>goto b x30</li>
<li>72C 2 min</li>
<li>16C hold</li>
</ol></ol>
<li>TBE Gel</li>
<ol type="A">
<li>Mix 120 uL TBE and 40 uL 6X dye</li>
<li>Aliquot 10 uL per sample on to parafilm</li>
<li>Add 2 uL sample or 1.5 uL ladder to aliquots</li>
<li>Mix and add 10 uL per lane to gel</li>
<li>Run gel at 230V for 24 minutes</li>
<li>Add 2 uL SYBR gold and incubate on shaker for 3 minutes</li>
<li>Rinse and image in gel doc</li></ol>
</ol>
 
 
<gallery perrow=3 heights=300px widths=300px>
File:20170501-qpCR-ProductionRun-PostBglii.png|qPCR curves (NEB amplified samples blue; TF amplified samples red)
File:2017-05-01-ProductionRun-PostBglii-NEB-NoAmp.png|NEB Gel
File:2017-05-01-ProductionRun-PostBglii-NEB-NoAmp.png|NEB Gel
File:2017-05-01-ProductionRun-PostBglii-TF-NoAmp.png|Thermo Fisher RCA
File:2017-05-01-ProductionRun-PostBglii-TF-NoAmp.png|Thermo Fisher RCA
File:2017-05-01-ProductionRun-PostBglii-NEB-PostAmp.png|NEB Gel-Post qPCR
File:2017-05-01-ProductionRun-PostBglii-TF-PostAmp.png|ThermoFisher Gel Post qPCR
</gallery>
</gallery>
Although the gel quality is poor, I do like these results, especially the pre-qPCR bands in the ThermoFisher lanes. I'm going to check the results via sequencing. See [[Daniel:Notebook/ComboLock/2017-5-2|tomorrow]].
[[Category:ComboLock]] [[Category:20170428]]

Latest revision as of 16:28, 2 May 2017

Production Run (Started Friday 4/28)[edit]

Back to Calendar

Protocol-Part 5[edit]

  1. qPCR
    1. Make 17.2X master mix according to following (1X)
      1. 361.2 uL nfH2O (21)
      2. 17.2 uL 10 uM AmpF6.4Sol (1)
      3. 430 uL 2X Kapa SYBR Fast Master Mix (25)
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout
    4. File:PlateLayout-20170501-ProductionRun-PostBglii.png
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix 120 uL TBE and 40 uL 6X dye
    2. Aliquot 10 uL per sample on to parafilm
    3. Add 2 uL sample or 1.5 uL ladder to aliquots
    4. Mix and add 10 uL per lane to gel
    5. Run gel at 230V for 24 minutes
    6. Add 2 uL SYBR gold and incubate on shaker for 3 minutes
    7. Rinse and image in gel doc


Although the gel quality is poor, I do like these results, especially the pre-qPCR bands in the ThermoFisher lanes. I'm going to check the results via sequencing. See tomorrow.