Daniel:Notebook/ComboLock/2017-2-27

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Revision as of 20:13, 27 February 2017 by >Djacobse (→‎Protocol)
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RCA + Circularization Test

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Repeating the circularization and RCA test using the three different polymerase enzymes to classify which is best to use. This will use the C4-C2 template, which is the simplified template that is the equivalent of the two C probe arms ligated and gap-filled.

Protocol

  1. Phosphorylation-Padlock0501
    1. Set up reaction according to table
    2. Reagent Stock Conc Final Conc./Amount uL added
      T4 Polynucleotide Kinase Buffer 10X 1X 2
      ATP 10 mM 1 mM 2
      PCCB-02 10 uM 100 pmol total 10
      T4 DNA Kinase 10 U/uL 10 U 1
      nfH2O NA NA 5
      Total     20
    3. Incubate at 37C for 30 min
    4. Heat kill enzyme with 15 minutes at 65C
  2. Template-Bead Binding
    1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Dilute 5 uL C4-C2 template into 45 uL nfH2O
    4. Add 2 uL diluted template per sample to bead solution; incubate at RT for 5 min
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  3. Padlock Hybridization
    1. Make 2 of the following master mix
      1. 8.2 uL 10 uM padlock0501
      2. 8.2 uL Amp Ligase 10X reaction buffer
      3. 65.6 uL nfH2O
    2. Resuspend sample in 20 uL 1X Amp Ligase buffer
    3. Incubate at 37C for 30 minutes
  4. Circularization
    1. Prepare 8.5X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (8.5x) (uL)
      NAD+ 5 mM 40 nmol 8 68
      dNTP 1 mM 600 pmol 0.6 5.1
      Betaine 5 M 15 umol 3 25.5
      10X AmpLigase Buffer 10X 1X 2 17
      Amp Ligase 5 U/uL 10 U 2 17
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 27.2
      nf H2O     1.2 8.4
      Total     20 140
    3. Add 20 uL phusion mix to 20 uL reaction
    4. Incubate at 40C for 4 hours

    Continued tomorrow