Daniel:Protocols/RCA

From ZhangLabWiki
Revision as of 20:44, 28 November 2016 by >Djacobse (→‎Protocol)
Jump to navigation Jump to search

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
    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. Incubate at 65C for 10 minutes