Daniel:Notebook/ComboLock/2017-3-6: Difference between revisions
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>Djacobse (Created page with "=Padlock0601 Test (Started Wed March 1)= Back to Calendar After reading some papers I'm going to give it...") |
>Djacobse |
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<li>Maximal Rolling Circle Amplification</li> | <li>Maximal Rolling Circle Amplification</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li> | <li>Create master mix according to following table</li> | ||
<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="180" height="30" | Reagent | |||
| width="85" | Single Rxn Vol | |||
| width="85" | 16.2X MM uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Template | |||
| align="center" align="center" valign="bottom" | 4 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | RCA Hexamer (1 mM) | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 8.1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (1 mM) | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 81 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 32.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | BSA (10 mg/mL) | |||
| align="center" align="center" valign="bottom" | 0.4 | |||
| align="center" align="center" valign="bottom" | 6.48 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 7.1 | |||
| align="center" align="center" valign="bottom" | 115.02 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 243 | |||
|} | |||
<li>Add 16 uL master mix to each tube</li> | |||
<li>Add 4 uL template according to table</li> | <li>Add 4 uL template according to table</li> | ||
<li>Incubate at 95 C for 5 minutes</li> | <li>Incubate at 95 C for 5 minutes</li> | ||
Line 20: | Line 69: | ||
<li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li> | <li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li> | ||
</ol></ol> | </ol></ol> | ||
[[Category:ComboLock]] [[Category:20170301]] |
Revision as of 00:16, 7 March 2017
Padlock0601 Test (Started Wed March 1)
After reading some papers I'm going to give it one more go to see if I can't amplify the DNA from last week better. I'll use the method of largest amplification I know: most time using random hexamers as primers.
Protocol
- Maximal Rolling Circle Amplification
- Create master mix according to following table
- Add 16 uL master mix to each tube
- Add 4 uL template 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 15 hours at 30C
- Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C
Reagent | Single Rxn Vol | 16.2X MM uL |
Template | 4 | 0 |
RCA Hexamer (1 mM) | 0.5 | 8.1 |
dNTP (1 mM) | 5 | 81 |
10X Buffer | 2 | 32.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 6.48 |
nfH2O | 7.1 | 115.02 |
Total | 20 | 243 |