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CUT&RUN Core Complete Set Accessory Reagents

This set contains CUT&RUN anti-DYKDDDDK antibody M, CUT&RUN Secondary, CUT&RUN Positive and Negative Control for the CUT&RUN method for improved genome-wide detection of Protein-DNA-Interactions.
Catalog No. ABIN6923131
$387.50
Plus shipping costs $45.00
24 tests
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  • Reactivity
    Eukaryotes
    Application
    Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag)
    Purpose
    This set contains CUT&RUN anti-DYKDDDDK antibody M, CUT&RUN Secondary, CUT&RUN Positive and Negative Control for the CUT&RUN method for improved genome-wide detection of Protein-DNA-Interactions.
    Characteristics
    CUT&RUN (Cleavage Under Targets And Release Using Nuclease) offers a new approach to pursue epigenetics.
    CUT&RUN overcomes various downfalls of ChIP-Seq with improved workflow.
    CUT&RUN-Sequencing has the advantage of being a simpler technique with lower costs due to the high signal-to-noise ratio, requiring less depth in sequencing.
    CUT&RUN has the potential to replace all ChIP-based applications.
    Components
    • CUT&RUN Positive Control (Recombinant Rabbit anti-H3K27me3 Antibody)
    • CUT&RUN Negative Control (Polyclonal Guinea Pig anti-Rabbit IgG Antibody, Pre-Adsorbed)
    • CUT&RUN anti-DYKDDDDK antibody M (Mouse anti-DYKDDDDK Tag Antibody)
    • CUT&RUN Secondary (Rabbit anti-Mouse IgG (H&L) Antibody)
    Material not included
  • Reagent Preparation
    • Wash Buffer
    • Binding Buffer
    • Antibody Buffer
    • Digitonin Wash Buffer
    • 2x Stop Buffer
    • Low Salt Rinse Buffer
    • Low Salt Incubation Buffer
    • Low Salt Stop Buffer
    Assay Procedure
    1. Cell Harvest

      • Harvest cells for each sample at RT
      • Wash cells 4 x with 1 mL Wash Buffer

    2. Prepare CUT&RUN Concanavalin A Beads

      • Pipette 10 µL CUT&RUN Concanavalin A Beads slurry for each sample into a tube
      • Place the tubes on a magnet separator and remove the liquid carefully
      • Remove the tubes from the magnetic separator
      • Wash beads 3 more times with 1 mL Binding Buffer
      • Finally resuspend the beads with 10 µL Binding Buffer per sample

    3. Cell Immobilization – binding to CUT&RUN Concanavalin A Beads

      • Carefully vortex the samples and add 10 µL of the prepared CUT&RUN Concanavalin A Beads to each sample
      • Close tubes tightly and rotate for 5-10 min at RT

    4. Cell Permeabilization and Primary Antibody Binding

      • Place the tubes on a magnetic separator and remove the liquid carefully
      • Remove the tubes from the magnetic separator
      • Place each tube on the vortex mixer set to a low speed and add 100 µL Antibody Buffer containing Digitonin
      • Gently vortex the tubes until the beads are resuspended
      • Add 5 µL CUT&RUN anti-DYKDDDDK antibody M or CUT&RUN Positive Control or CUT&RUN Negative Control corresponding to a 1:20 dilution
      • Add 1 µL primary rabbit antibody against your protein of interest corresponding to a 1:100 dilution to all samples
      • Rotate the tubes for 2 h at RT or 4 h to O/N at 4 °C
      • Place the tubes on a magnet separator and remove the liquid carefully
      • Remove the tubes from the magnetic separator
      • Resuspend pellet with 1 mL Digitonin Wash Buffer and mix by inversion
      • Wash again

    5. Secondary Antibody Binding (optional)
       If no secondary antibody is used proceed directly to pA/G-MNase-Binding.

      • Place the tubes on a magnet separator and remove the liquid carefully
      • Remove the tubes from the magnetic separator
      • Vortex the samples at low speed and add 100 µL Digitonin Wash Buffer per sample
      • Add 5 µL CUT&RUN Secondary Antibody corresponding to a 1:20 dilution
      • Rotate the tubes for 1 h at 4 °C
      • Place the tubes on a magnet separator and remove the liquid carefully
      • Remove the tubes from the magnetic separator
      • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion
      • Wash again

    6. Protein A-MNase or Protein A/G-MNase Binding

      • Place the tubes on a magnet separator and remove the liquid carefully
      • Remove the tubes from the magnetic separator
      • Place each tube on the vortex mixer set to a low speed and add 50 µL Digitonin Wash Buffer and 2.5 µL pA/G-MNase per sample
      • Rotate the tubes for 1 h at 4 °C
      • Place the tubes on a magnet separator and remove the liquid carefully
      • Remove the tubes from the magnetic separator
      • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion
      • Wash again

    7. MNase Digestion and Release of pA/G-MNase-Bound Chromatin Fragments

      High Ca2+/Low Salt Chromatin Cleavage

      • Quick pulse in a table-top centrifuge (max 100 x g)
      • Place the tubes on a magnet separator and remove the liquid carefully
      • Resuspend with 1 mL Low-Salt Rinse Buffer and mix by inversion
      • Quick pulse in a table-top centrifuge (max 100 x g)
      • Place the tubes on a magnet separator and remove the liquid carefully
      • Wash again
      • Place each tube on the vortex mixer set to a low speed and add 200 µL ice cold Low Salt Incubation Buffer per sample
      • Incubate tubes at 0 °C for 5 min
      • Place the tubes on a cold magnet separator and remove the liquid carefully
      • Remove the tubes from the magnetic separator
      • Resuspend with 200 µL Low Salt Stop Buffer and mix by gentle vortexing
      • Incubate tubes at 37 °C for 30 min
      • Place the tubes on a magnet separator
      • Transfer the supernatant containing the pA/G-MNase-bound digested chromatin fragments to fresh 1.5 mL tubes
      • Proceed with DNA extraction
    Restrictions
    For Research Use only
  • Buffer
    CUT&RUN Positive Control: 50 % Glycerol/PBS, 1 % BSA, 0.09 % (w/v) Sodium Azide
    CUT&RUN Negative Control & CUT&RUN anti-DYKDDDDK antibody M & CUT&RUN Secondary: 0.02 M Potassium Phosphate, 0.15 M NaCl, pH 7.2, 0.01 % (w/v) Sodium Azide
    Preservative
    Sodium azide
    Precaution of Use
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Storage
    4 °C,-20 °C
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