Daniel:Notebook/ComboLock/2017-3-27: Difference between revisions
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>Djacobse (Created page with "=RCA Test (Started March 24)= Back to Calendar ==Protocol== <ol> <li>Rolling Circle Amplification</li>...") |
>Djacobse |
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<li>Add 48 uL master mix to each well</li> | |||
<li>Add 2 uL sample according to plate layout</li> | |||
[[Image:PlateLayout-20170323-CPProduction.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>Qubit Measurement</li> | |||
<ol type="A"> | |||
<li>Add 995 uL buffer and 5 uL ssdye to tube</li> | |||
<li>Add 190 uL buffer and 10 uL correct standard to standard tubes</li> | |||
<li>Add 199 uL buffer and 1 uL sample to sample tubes</li> | |||
<li>Let sit 5-10 minutes in the dark</li> | |||
<li>Measure in Qubit</li></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> | |||
===Results=== | |||
<gallery perrow=3 heights=300px widths=300px> | |||
File:|qPCR curves | |||
File:|Circular Product target (104bp) | |||
File:|AmpF/AmpR (221 bp) | |||
</gallery> |
Revision as of 16:16, 27 March 2017
RCA Test (Started March 24)
Protocol
- Rolling Circle Amplification
- Add 20 uL nfH2O to every sample
- qPCR
- Make master mixes according to following table
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170323-CPProduction.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
- Qubit Measurement
- Add 995 uL buffer and 5 uL ssdye to tube
- Add 190 uL buffer and 10 uL correct standard to standard tubes
- Add 199 uL buffer and 1 uL sample to sample tubes
- Let sit 5-10 minutes in the dark
- Measure in Qubit
- 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
Reagent | 1X Rxn | MM-Amp (33.2X) | MM-Prod (9.2X) |
Primer Pair | NA | AmpF/AmpR | p4/p12RC |
Forward Primer (10 uM) | 1 | 33.2 | 9.2 |
Forward Primer (10 uM) | 1 | 33.2 | 9.2 |
Kapa SYBR Fast Master Mix (2X) | 25 | 830 | 230 |
nfH2O | 21 | 697.2 | 193.2 |
Total | 48 | 1593.6 | 441.6 |