Daniel:Notebook/ComboLock/2017-4-12: Difference between revisions
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>Djacobse |
>Djacobse |
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<li>Incubate 2 hr 37C</li> | <li>Incubate 2 hr 37C</li> | ||
<li>Heat kill with 65C for 20 min</li></ol> | <li>Heat kill with 65C for 20 min</li></ol> | ||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 120 uL 10X TBE and 40 uL 6X dye</li> | |||
<li>Aliquot 8 uL TBE/dye mix onto parafilm per sample</li> | |||
<li>Add 4 uL sample to sample lanes</li> | |||
<li>Mix 8 uL 1X TBE, 2 uL 6X dye, and 1.5 uL 25bp ladder for ladder sample</li> | |||
<li>Add 10 uL from aliquots into appropriate gel lanes</li> | |||
<li>Run gel for 25 minutes at 250V</li> | |||
<li>Stain with 2 uL SYBR gold for 3 min</li> | |||
<li>Rinse and image in gel doc</li></ol> | |||
</ol> | </ol> | ||
<gallery perrow=2 heights=300px widths=300px> | |||
File:| | |||
File:| | |||
</gallery> |
Revision as of 20:00, 12 April 2017
RCA Double Deuce
Protocol
- 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
- TBE Gel
- Mix 120 uL 10X TBE and 40 uL 6X dye
- Aliquot 8 uL TBE/dye mix onto parafilm per sample
- Add 4 uL sample to sample lanes
- Mix 8 uL 1X TBE, 2 uL 6X dye, and 1.5 uL 25bp ladder for ladder sample
- Add 10 uL from aliquots into appropriate gel lanes
- Run gel for 25 minutes at 250V
- Stain with 2 uL SYBR gold for 3 min
- Rinse and image in gel doc
Reagent | Single Rxn Vol | Master Mix (13.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 26.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 132 |
Total | 20 | 158.4 |