Daniel:Notebook/ComboLock/2017-7-17: Difference between revisions
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<li>Heat kill with 65C for 20 min</li> | <li>Heat kill with 65C for 20 min</li> | ||
<li>Hold at 12C</ol> | <li>Hold at 12C</ol> | ||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make 5.2X master mix according to following (1X)</li> | |||
<ol type="a"> | |||
<li>109.2 uL nfH<sub>2</sub>O (21)</li> | |||
<li>5.2 uL 10 uM AmpF6.4Sol (1)</li> | |||
<li>130 uL 2X Kapa SYBR Fast Master Mix (25)</li></ol> | |||
<li>Add 47 uL master mix to each well</li> | |||
<li>Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout</li> | |||
[[Image:PlateLayout20170717-MultiplexExperiment.png|250x350px]] | |||
<li>Run the [[Daniel:Protocols/qPCR|Sstandard qPCR protocol]] for ~30 cycles</li> | |||
</ol> | |||
<li>TBE Gel</li> | |||
<li>Mix together 64 uL TBE and 16 uL 6X dye</li> | |||
<li>Aliquot 10 uL/lane onto parafilm</li> | |||
<li>Add 2 uL correct sample or 1.5 uL 25bp ladder to aliquots</li> | |||
<li>Add 10 uL aliquots to correct lanes</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>Image in gel doc</li> | |||
</ol> | </ol> |
Revision as of 16:02, 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
- 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 |