Daniel:Notebook/ComboLock/2017-6-6: Difference between revisions
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<li>Hold at 12C</ol> | <li>Hold at 12C</ol> | ||
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Continued [[Daniel:Notebook/ComboLock/2017-6-7|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20170605]] | [[Category:ComboLock]] [[Category:20170605]] |
Latest revision as of 17:11, 7 June 2017
Sensitivity and Specificity Experiments (Started Yesterday)[edit]
Protocol-Part 4[edit]
- Exonuclease Digestion
- Mix 17.5 uL Exo I (20U/uL) and 3.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 2 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 15 uL master mix to each tube
- Add 4 uL sample to appropriate tubes
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add 1 uL enzyme
- Incubate for 3 hours at 30C
- Incubate for 65 minutes at 20C to denature
- 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
Reagent | Single Rxn (uL) | Master Mix X (10.2X) |
Enzyme | NA | Thermo |
Sample | 4 | 0 |
p12RC*** (10 uM) | 0.2 | 2.04 |
RCA Primer LLRC*** (10 uM) | 0.2 | 2.04 |
dNTP (10 mM) | 0.5 | 5.1 |
10X Buffer | 2 | 20.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 4.08 |
nfH2O | 11.7 | 119.34 |
Total | 20 | 153 |
Reagent | Single Rxn Vol | Master Mix (10.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 20.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 102 |
Total | 20 | 122.4 |
Continued tomorrow