Daniel:Notebook/ComboLock/2016-7-29: Difference between revisions
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File:2016-07-29-PhusionTest-RawSample-Capture2.png|Raw Sample Gel Image (TBU) | File:2016-07-29-PhusionTest-RawSample-Capture2.png|Raw Sample Gel Image (TBU) | ||
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[[Category:ComboLock]] [[Category:20160728]] |
Latest revision as of 00:15, 18 August 2016
Phusion Test (Started yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Chill on ice 1 min
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 1 uL exonuclease I (20 units/μL) and 1 uL exonuclease III (100 units/μL) per sample
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Set up reaction according to table
- Master Mix
- 152.5 uL KAPA SyberFast MM
- 103.7 uL nfH2O
- 3.05 uL AmpF 6.4 100 uM
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- 2016-07-29-qPCR-PhusionTest.png
qPCR Curve
- 2016-07-29-PhusionTest.png
Gel Image
- 2016-07-29-PhusionTest-RawSample.png
Raw Sample Gel Image (TBU)
- 2016-07-29-PhusionTest-RawSample-Capture2.png
Raw Sample Gel Image (TBU)
Reagent | Stock Concentration | Reagent Added | uL Added per sample | Master Mix (5.1X) |
ExoI | 20U/uL | 20 | 1 | 5.1 |
ExoIII | 100 U/uL | 100 | 1 | 5.1 |
Sample | Lane | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 (Hs-Hemo Klentaq) | A1 | 15 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 2 (Hs-Phusion) | B1 | 16 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 3 (Hs-No C-Probe) | C1 | 17 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 4 (mm-Phusion) | D1 | 18 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 5 (NTC) | E1 | 19 | 2.5 | 25 | 0.5 | 5 | 17 | 50 |
Sample 6 (No Sample) | F1 | 20 | 2.5 | 25 | 0.5 | 5 | 19 | 50 |