CUT&RUN Core Set

Produktnummer ABIN6923134

Produktdetails

Reaktivität: Eukaryonten

Applikation: Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag)

Verwendungszweck: This set contains CUT&RUN Positive and Negative Control for the CUT&RUN method for improved genome-wide detection of Protein-DNA-Interactions.

Produktmerkmale: 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.

Bestandteile:

• CUT&RUN Positive Control (Recombinant Rabbit anti-H3K27me3 Antibody)
• CUT&RUN Negative Control (Polyclonal Guinea Pig anti-Rabbit IgG Antibody, Pre-Adsorbed)

Benötigtes Material:

• Specific antibody against target of interest
• pA/G-MNase (ABIN6950951)
• CUT&RUN Concanavalin A Beads (ABIN6952467)

Anwendungsinformationen

Aufbereitung der Reagenzien:

• Wash Buffer
• Binding Buffer
• Antibody Buffer
• Digitonin Wash Buffer
• 2x Stop Buffer
• Low Salt Rinse Buffer
• Low Salt Incubation Buffer
• Low Salt Stop Buffer

Testdurchführung:

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 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 the remaining 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 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

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Buffer: CUT&RUN Positive Control: 50 % Glycerol/PBS, 1 % BSA, 0.09 % (w/v) Sodium Azide
CUT&RUN Negative Control: 0.02 M Potassium Phosphate, 0.15 M NaCl, pH 7.2, 0.01 % (w/v) Sodium Azide

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Lagerung: 4 °C