Daniel:Notebook/ComboLock/2017-6-7: Difference between revisions
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>Djacobse (Created page with "=Sensitivity and Specificity Experiments (Started Monday)= Back to Calendar ==Protocol== <ol start="10"...") |
>Djacobse |
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Line 28: | Line 28: | ||
<li>72C 2 min</li> | <li>72C 2 min</li> | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol></ol> | |||
<li>TBE Gel</li> | |||
<li>Mix together 120 uL TBE and 30 uL 6X dye</li> | |||
<li>Aliquot 10 uL/lane onto parafilm</li> | |||
<li>Add 2 uL correct sample or (1.5 uL for ladder) to aliquots</li> | |||
<li>Add 10 uL aliquots to correct lanes</li> | |||
<li>Run gel at 235V for 24 minutes</li> | |||
<li>Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes</li> | |||
<li>Image in gel doc</li> | |||
</ol> | |||
==Results== | |||
<gallery perrow=2 heights=250px widths=350px mode=packed-hover> | |||
File:PlateLayout-SensSpec-20170607.png|Plate Layout | |||
File:SensSpec-PostBglii-20170607-plateCTs.png|Raw CT values | |||
File:SensSpec-PostBglii-20170607-plateCTs-normed.png|CT values normalized to NTC (Lane H3) | |||
File:SensSpec-PostBglii-20170607-rawcurves.png|Raw curves | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20170605]] | [[Category:ComboLock]] [[Category:20170605]] |
Revision as of 17:11, 7 June 2017
Sensitivity and Specificity Experiments (Started Monday)
Protocol
- 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-SensSpec-20170607.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 together 120 uL TBE and 30 uL 6X dye
- Aliquot 10 uL/lane onto parafilm
- Add 2 uL correct sample or (1.5 uL for ladder) to aliquots
- Add 10 uL aliquots to correct lanes
- Run gel at 235V for 24 minutes
- Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes
- Image in gel doc
Results
- PlateLayout-SensSpec-20170607.png
Plate Layout
- SensSpec-PostBglii-20170607-plateCTs.png
Raw CT values
- SensSpec-PostBglii-20170607-plateCTs-normed.png
CT values normalized to NTC (Lane H3)
- SensSpec-PostBglii-20170607-rawcurves.png
Raw curves