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" | &nbsp;
| align="center" align="center" | &nbsp;
| 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" | &nbsp;
| align="center" align="center" | &nbsp;
| 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

Back to Main

Protocol page for basic circularization of padlock probes and rolling circle amplification of the finished product. Includes exonuclease digestion to get rid of

Protocol

  1. Circularization
    1. Prepare (N+1)X uL Phusion mix (N samples)
    2. 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
    3. Add 20 uL Phusion mix to 20 uL of reaction suspended in 1X buffer
    4. Incubate at 60C for 2 hours
    5. Heat kill the reaction by incubating for 2 minutes at 94C
  2. Exonuclease Digestion