Daniel:Notebook/ComboLock/2017-3-2: Difference between revisions
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>Djacobse (Created page with "=Padlock0601 Test (Started Yesterday)= Back to Calendar ==Protocol-Part 3== <ol start="6"> <li>Exonucle...") |
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<li>Rolling Circle Amplification</li> | <li>Rolling Circle Amplification</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Samples will be aliquoted under following rules</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |||
| width="200" height="30" | Samples | |||
| width="220" | Condition | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample X (1-4, AB) | |||
| align="center" valign="bottom" | Unpurified | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample Y (1-4, AB) | |||
| align="center" valign="bottom" | Purified | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | A Samples (1-4) | |||
| align="center" valign="bottom" | Normal Primer | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | B Samples (1-4) | |||
| align="center" valign="bottom" | Phosphorothiorate Primer | |||
|} | |||
<li>Prepare 8.2X Master mix according to table below</li> | <li>Prepare 8.2X Master mix according to table below</li> | ||
<li>Add 15 uL master mix to each tube</li> | <li>Add 15 uL master mix to each tube</li> |
Revision as of 17:19, 2 March 2017
Padlock0601 Test (Started Yesterday)
Protocol-Part 3
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Column Purification-Qiaquick
- Add 150 uL (5X) PB (binding buffer) to 30 uL sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;
- Repeat wash step;
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Elute with 30 uL EB
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- Rolling Circle Amplification
- Samples will be aliquoted under following rules
- Prepare 8.2X Master mix according to table below
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table
Samples | Condition |
Sample X (1-4, AB) | Unpurified |
Sample Y (1-4, AB) | Purified |
A Samples (1-4) | Normal Primer |
B Samples (1-4) | Phosphorothiorate Primer |