Daniel:Notebook/ComboLock/2017-3-2: Difference between revisions
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>Djacobse |
>Djacobse |
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<li>Prepare 8.2X Master mix according to table below</li> | <li>Prepare 8.2X Master mix according to table below</li> | ||
<li>Add | |||
<li>Add | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | |||
| width="160" height="30" | Reagent | |||
| width="85" | Single Rxn Vol | |||
| width="85" | 16.2X MM uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Template | |||
| align="right" align="center" valign="bottom" | 4 | |||
| align="right" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | RCA Primer (10 uM) | |||
| align="right" align="center" valign="bottom" | 2.5 | |||
| align="right" align="center" valign="bottom" | 0 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (1 mM) | |||
| align="right" align="center" valign="bottom" | 5 | |||
| align="right" align="center" valign="bottom" | 81 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | |||
| align="right" align="center" valign="bottom" | 2 | |||
| align="right" align="center" valign="bottom" | 32.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="right" align="center" valign="bottom" | 1 | |||
| align="right" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | BSA (10 mg/mL) | |||
| align="right" align="center" valign="bottom" | 0.4 | |||
| align="right" align="center" valign="bottom" | 6.48 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="right" align="center" valign="bottom" | 5.1 | |||
| align="right" align="center" valign="bottom" | 82.62 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="right" align="center" valign="bottom" | 20 | |||
|style="font-weight:bold" align="right" align="center" valign="bottom" | 202.5 | |||
|} | |||
<li>Add 12.5 uL master mix to each tube</li> | |||
<li>Add 4 uL template according to table</li> | |||
<li>Add 2.5 uL correct primer according to table</li> | |||
<li>Incubate at 95 C for 5 minutes</li> | |||
<li>Lower to 55C for 15 minutes</li> | |||
<li>Lower to 30C for at least 2 minutes</li> | |||
<li>Add 1 uL Phi29 polymerase to each sample</li> | |||
<li>Incubate 3 hours at 30C</li> | |||
<li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li> | |||
</ol></ol> | |||
==Protocol-Part 4== | |||
<ol start="10"> | |||
</ol> | |||
[[Category:ComboLock]] [[Category:20170301]] | [[Category:ComboLock]] [[Category:20170301]] |
Revision as of 17:26, 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 12.5 uL master mix to each tube
- Add 4 uL template according to table
- Add 2.5 uL correct primer according to table
- Incubate at 95 C for 5 minutes
- Lower to 55C for 15 minutes
- Lower to 30C for at least 2 minutes
- Add 1 uL Phi29 polymerase to each sample
- Incubate 3 hours at 30C
- Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C
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 |
Reagent | Single Rxn Vol | 16.2X MM uL |
Template | 4 | 0 |
RCA Primer (10 uM) | 2.5 | 0 |
dNTP (1 mM) | 5 | 81 |
10X Buffer | 2 | 32.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 6.48 |
nfH2O | 5.1 | 82.62 |
Total | 20 | 202.5 |