Daniel:Protocols/RCA: Difference between revisions

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<li>Add 5 uL sample to correct reaction tubes</li>
<li>Add 5 uL sample to correct reaction tubes</li>
<li>Incubate at 37C for 3 hours</li>
<li>Incubate at 37C for 3 hours</li>
<li>Incubate at 65C for 10 minutes</li></ol>
<li>Heat kill by incubating at 65C for 10 minutes</li></ol>
</ol>
 
 
</ol>
</ol>

Latest revision as of 22:27, 28 November 2016

Circularization and Rolling Circle Amplification[edit]

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

Protocol[edit]

  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
    1. Combine equal molar amounts of Exonuclease I (20U/uL) and Exonuclease III (100U/uL)
    2. Add 2 uL of mix to each reaction
    3. Incubate for 30 minutes to 2 hours at 37C
    4. Heat kill by incubating for 5 minutes at 94C
  3. Rolling Circle Amplification
    1. Prepare (N+1)X master mix according to table below
    2. Reagent Stock Concentration Final Conc/Amt uL Added in 20 uL Rxn
      Template NA NA 5
      RCA Primer 10 uM 2 uM 4
      dNTP 1 mM 50 uM 1
      10X Buffer 10X 1X 2
      Phi29 (NEB) 10 U/uL 10 U 1
      nfH2O NA NA 7
      Total     20
    3. Add 15 uL master mix to each tube
    4. Add 5 uL sample to correct reaction tubes
    5. Incubate at 37C for 3 hours
    6. Heat kill by incubating at 65C for 10 minutes