General Protocols for Downstream Applications (NEB #E1603)
Sanger Sequencing Sample Preparation
- Dilute RCA products 5- to 20-fold with nuclease-free water.
- 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
- Dilute RCA products 20-fold with nuclease-free water.
- 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.
- Dilute RCA products two-fold with nuclease-free water.
- Purify RCA products using 0.6X SPRI beads following manufacturer’s recommendations.
- Prepare debranching reactions as described below. Mix well by pipetting, and centrifuge briefly to collect solutions to the bottom of the tube.
30 µl REACTION
NEBuffer 2, 10X
T7 Endonuclease I (NEB #M0302)
to 30 µl
- Incubate for 1 hour at 37°C. Debranched product can be further purified by SPRI beads cleanup following manufacturer’s protocol.