Daniel:Notebook/ComboLock/2017-4-4: Difference between revisions
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>Djacobse |
>Djacobse |
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Line 48: | Line 48: | ||
<li>Ramp to 50C at 0.2C/s</li> | <li>Ramp to 50C at 0.2C/s</li> | ||
<li>10 min 50C</li> | <li>10 min 50C</li> | ||
<li> | <li>4 hr 37C; '''When this cycle starts add 2 uL Bglii'''</li> | ||
<li>Heat kill with 65C for 20 min</li></ol> | <li>Heat kill with 65C for 20 min</li></ol> | ||
</ol> | </ol> |
Revision as of 17:25, 4 April 2017
RCA Test (Started March 24)
Bgl Digestion-Excessive
- Bgl Digestion
- Make the following reactions; Do not add Bglii yet
- Use the following thermocycler program
- 5 min 95C
- Ramp to 50C at 0.2C/s
- 10 min 50C
- 4 hr 37C; When this cycle starts add 2 uL Bglii
- Heat kill with 65C for 20 min
- TBE Gel
- Mix 60 uL 10X TBE and 20 uL 6X dye
- Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot
- Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
Reagent | Single Rxn Vol | Master Mix (6.2X) |
RCA Rxn | 10 | 0 |
10X Buffer 3.1 | 4 | 24.8 |
Bglii | 2 | 0 |
nfH2O | 24 | 148.8 |
Total | 40 | 173.6 |