Daniel:Notebook/ComboLock/2017-3-27: Difference between revisions
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File:2017-03-27-RCATest-CPLP-NEB-12hr.png|Gel image-NEB-12hr | File:2017-03-27-RCATest-CPLP-NEB-12hr.png|Gel image-NEB-12hr | ||
File:2017-03-27-RCATest-CPLP-NEB-adapter.png|Gel image-NEB-3hr-adapters | File:2017-03-27-RCATest-CPLP-NEB-adapter.png|Gel image-NEB-3hr-adapters | ||
</gallery> | |||
===Discussion=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:20170327-RCATest-CPLP-CT-EnzymeComparison.png|Comparing the three enzymes (3 hrs RCA) | |||
File:20170327-RCATest-CPLP-CT-TimeComparison.png|Comparing CT values between RCA times (NEB) | |||
</gallery> | </gallery> | ||
[[Category:ComboLock]] [[Category:20170324]] | [[Category:ComboLock]] [[Category:20170324]] |
Revision as of 17:12, 3 April 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-20170327-RCATest-CPLP.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 |
Results
- 20170327-qPCR-RCATest-CPLP-EnzymeComparison.png
qPCR curves showing enzyme comparison (All 3hrs)
- 20170327-qPCR-RCATest-CPLP-TimeComparison.png
qPCR curves showing RCA Time comparison (NEB)
- 20170327-qPCR-RCATest-CPLP-AverageComparison.png
qPCR curves showing Averaged comparisons (All conditions)
- 2017-03-27-RCATest-CPLP-NEB-3hr.png
Gel image-NEB-3hr
- 2017-03-27-RCATest-CPLP-TF-3hr.png
Gel image-TF-3hr
- 2017-03-27-RCATest-CPLP-Epi-3hr.png
gel image-Epi-3hr
- 2017-03-27-RCATest-CPLP-NEB-6hr.png
Gel image-NEB-6hr
- 2017-03-27-RCATest-CPLP-NEB-12hr.png
Gel image-NEB-12hr
- 2017-03-27-RCATest-CPLP-NEB-adapter.png
Gel image-NEB-3hr-adapters
Discussion
- 20170327-RCATest-CPLP-CT-EnzymeComparison.png
Comparing the three enzymes (3 hrs RCA)
- 20170327-RCATest-CPLP-CT-TimeComparison.png
Comparing CT values between RCA times (NEB)