Home Protocols General Protocols for Downstream Applications (NEB #E1603)

General Protocols for Downstream Applications (NEB #E1603)

Sanger Sequencing Sample Preparation 

  1. Dilute RCA products 5- to 20-fold with nuclease-free water.

  2. Use 1 µl of diluted product to prepare Sanger sequencing samples according to sequence provider’s instructions.

Cell-free Protein Expression from RCA Products Protocol

  1. Dilute RCA products 20-fold with nuclease-free water.

  2. Use 12 µl diluted RCA products in a 50 µl NEBExpress® Cell-free E. coli Protein Synthesis System (NEB #E5360) reaction, as directed in the product manual.

Note: Protein expression can be verified by mixing 2 µl of the expression reaction with 6 µl Blue Protein Loading Dye (NEB #B7703) and 10 µl water followed by SDS-PAGE.


Debranching Protocol

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

  2. Purify RCA 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

    PurifiedRCAproducts

    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.