Home Protocols General Protocols for Downstream Applications (NEB #E1604)

General Protocols for Downstream Applications (NEB #E1604)

Debranching Protocol

  1. Dilute WGA products two-fold with nuclease-free water.

  2. Purify WGA products using 0.6X SPRI beads following manufacturer’s recommendations.

  3.  Prepare debranching reactions as described below. Mix well by pipetting, and centrifuge briefly to collect solutions to the bottom of the tube. 
       

    COMPONENTS

    30 µl REACTION

    FINAL CONCENTRATION

    Purified WGA products

    Variable

    Variable

    NEBuffer 2, 10X

    3 µl

    1X

    T7 Endonuclease I (NEB #M0302)

    1.5 µl

    		 0.5 units/µl

    Nuclease-free water

    to 30 µl

    N/A


  4. Incubate for 1 hour at 37°C. Debranched product can be further purified by SPRI beads cleanup following manufacturer’s protocol.

 

Next-generation sequencing Library Preparation Protocol

  1. Dilute WGA products with nuclease-free water (typically ~50-fold), and measure the concentration of the WGA products by Quant-iT® PicoGreen® dsDNA Assay Kit or Qubit® Fluorometer.

  2. Proceed with NGS library prep using the appropriate amount of WGA product. Assemble library according to manufacturer’s protocol.

    Note: If proceeding with Illumina sequencing, we recommend using the NEBNext® Ultra™ II FS Library Prep Kit (NEB #E7805).