Daniel:Notebook/ComboLock/2017-6-16
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Antibody Detection Test-Positive Control (Started Wednesday)[edit]
Protocol-Part 5 (From Yesterday)[edit]
- Bglii digestion
- Make the following reactions; Do not add Bglii yet
- Aliquot 12 uL master mix into new tube
- Add 7 uL appropriate sample
- Mix with vortexer, spin down, and add 1 uL Bglii
- Incubate 2 hr 37C
- Heat kill with 65C for 20 min
- Hold at 12C
- qPCR
- Make the following 7.2X master mix (1X)
- 7.2 uL 10 uM AmpF6.4Sol
- 151.2 uL (21) nfH2O
- 180 uL 2X Kapa SYBR Fast Master Mix
- Add 47 uL master mix to each lane
- Add 1 uL AmpR and 2 uL sample according to plate layout File:PlateLayout-20170616-AntibodyTest.png
- Use the following protocol
- 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
Reagent | Single Rxn Vol | Master Mix (6.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 12.4 |
Bglii | 1 | 6.2 |
nfH2O | 10 | 62 |
Total | 20 | 80.6 |
Results[edit]
- PlateLayout-20170616-AntibodyTest.png
Plate layout
- Antibodytest-controlsystem-20170615-plateCTs-normed.png
Plate CTs
- Antibodytest-controlsystem-20170615-rawcurves.png
Raw curves
- 2017-06-19-AntibodyTest-ControlSystem.png
Gel image