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- | =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= | ==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]
Protocol-Part 5[edit]
- qPCR
- Make 17.2X master mix according to following (1X)
- 361.2 uL nfH2O (21)
- 17.2 uL 10 uM AmpF6.4Sol (1)
- 430 uL 2X Kapa SYBR Fast Master Mix (25)
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout File:PlateLayout-20170501-ProductionRun-PostBglii.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 120 uL TBE and 40 uL 6X dye
- Aliquot 10 uL per sample on to parafilm
- Add 2 uL sample or 1.5 uL ladder to aliquots
- Mix and add 10 uL per lane to gel
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc
- 20170501-qpCR-ProductionRun-PostBglii.png
qPCR curves (NEB amplified samples blue; TF amplified samples red)
- 2017-05-01-ProductionRun-PostBglii-NEB-NoAmp.png
NEB Gel
- 2017-05-01-ProductionRun-PostBglii-TF-NoAmp.png
Thermo Fisher RCA
- 2017-05-01-ProductionRun-PostBglii-NEB-PostAmp.png
NEB Gel-Post qPCR
- 2017-05-01-ProductionRun-PostBglii-TF-PostAmp.png
ThermoFisher Gel Post qPCR
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.