Daniel:Notebook/ComboLock/2017-7-17: Difference between revisions
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>Djacobse |
>Djacobse |
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Line 64: | Line 64: | ||
</ol> | </ol> | ||
<li>TBE Gel</li> | <li>TBE Gel</li> | ||
<ol type="A" | |||
<li>Mix together 64 uL TBE and 16 uL 6X dye</li> | <li>Mix together 64 uL TBE and 16 uL 6X dye</li> | ||
<li>Aliquot 10 uL/lane onto parafilm</li> | <li>Aliquot 10 uL/lane onto parafilm</li> | ||
Line 70: | Line 71: | ||
<li>Run gel at 235V for 24 minutes</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>Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes</li> | ||
<li>Image in gel doc</li> | <li>Image in gel doc</li></ol> | ||
</ol> | </ol> | ||
==Results== | |||
<gallery perrow=2 heights=250px widths=350px mode=packed-hover> | |||
File:PlateLayout20170717-MultiplexExperiment.png|Plate layout | |||
File:|Plate CTs | |||
File:|Raw curves | |||
File:|Gel image | |||
</gallery> |
Revision as of 16:03, 17 July 2017
Multiplex Experiment (Started Thursday July 13)
Protocol-Part 4 (Continued from Friday July 14)
- 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 5.2X master mix according to following (1X)
- 109.2 uL nfH2O (21)
- 5.2 uL 10 uM AmpF6.4Sol (1)
- 130 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:PlateLayout20170717-MultiplexExperiment.png
- Run the Sstandard qPCR protocol for ~30 cycles
- TBE Gel <ol type="A"
- Mix together 64 uL TBE and 16 uL 6X dye
- Aliquot 10 uL/lane onto parafilm
- Add 2 uL correct sample or 1.5 uL 25bp 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 (4.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 8.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 42 |
Total | 20 | 50.4 |
Results
- PlateLayout20170717-MultiplexExperiment.png
Plate layout