Daniel:Protocols/RCA: Difference between revisions
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>Djacobse (Created page with "=Circularization and Rolling Circle Amplification= Back to Main Protocol page for basic circularization of padlock probes and ==Protocol==") |
>Djacobse |
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[[Daniel Jacobsen|Back to Main]] | [[Daniel Jacobsen|Back to Main]] | ||
Protocol page for basic circularization of padlock probes and | Protocol page for basic circularization of padlock probes and rolling circle amplification of the finished product. Includes exonuclease digestion to get rid of | ||
==Protocol== | ==Protocol== | ||
<ol> | |||
<li>Circularization</li> | |||
<ol type ="A"> | |||
<li>Prepare (N+1)X uL Phusion mix (N samples)</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="140" height="43" | Reagent | |||
| width="85" | Stock Conc | |||
| width="85" | Final Amount | |||
| width="120" | 1x Vol (uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | NAD+ | |||
| align="center" | 5 mM | |||
| align="center" | 40 nmol | |||
| align="center" align="center" | 8 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | dNTP | |||
| align="center" | 1 mM | |||
| align="center" | 600 pmol | |||
| align="center" align="center" | 0.6 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Betaine | |||
| align="center" | 5 M | |||
| align="center" | 15 umol | |||
| align="center" align="center" | 3 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X AmpLigase Buffer | |||
| align="center" | 10X | |||
| align="center" | 1X | |||
| align="center" align="center" | 2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase | |||
| align="center" | 5 U/uL | |||
| align="center" | 10 U | |||
| align="center" align="center" | 2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" valign="bottom" | Phusion HF DNA Polymerase | |||
| align="center" | 2000 U/mL | |||
| align="center" | 6.4U | |||
| align="center" align="center" | 3.2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nf H2O | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" align="center" | 1.2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Total | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
| align="center" align="center" | 20 | |||
|} | |||
<li>Add 20 uL Phusion mix to 20 uL of reaction suspended in 1X buffer</li> | |||
<li>Incubate at 60C for 2 hours</li> | |||
<li>Heat kill the reaction by incubating for 2 minutes at 94C</li></ol> | |||
<li>Exonuclease Digestion</li> | |||
</ol> |
Revision as of 20:35, 28 November 2016
Circularization and Rolling Circle Amplification
Protocol page for basic circularization of padlock probes and rolling circle amplification of the finished product. Includes exonuclease digestion to get rid of
Protocol
- Circularization
- Prepare (N+1)X uL Phusion mix (N samples)
- Add 20 uL Phusion mix to 20 uL of reaction suspended in 1X buffer
- Incubate at 60C for 2 hours
- Heat kill the reaction by incubating for 2 minutes at 94C
- Exonuclease Digestion
Reagent | Stock Conc | Final Amount | 1x Vol (uL) |
NAD+ | 5 mM | 40 nmol | 8 |
dNTP | 1 mM | 600 pmol | 0.6 |
Betaine | 5 M | 15 umol | 3 |
10X AmpLigase Buffer | 10X | 1X | 2 |
Amp Ligase | 5 U/uL | 10 U | 2 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 |
nf H2O | 1.2 | ||
Total | 20 |