Question

What does the enzyme exonuclease I do? Why did you treat your initial PCR product with exonuclease I before using the DNA as a template for Nested PCR? What would have been the result if you had not done an exonuclease I treatment?

Answer 1

ANSWERS

Enzymatic PCR cleanup method offers an easy way to remove the remaining primers and dNTP left from a PCR reaction. Two enzymes are needed to complete the process: Exonuclease I (Exo I, NEB #M0293) which degrades the residual PCR primers, and Shrimp Alkaline Phosphatase (rSAP, NEB #M0371) which dephosphorylates the remaining dNTP. This method enables direct downstream applications, such as Sanger sequencing, NGS, genotyping, SNP analysis and nested PCR etc. The two enzymes are added directly to the PCR reaction after thermal cycling, without changing buffer condition or additional additives. Further, these enzymes are 100% compatible with all commonly used PCR reaction buffers.

Protocol

1. Add 0.5 µl of Exo I and 1 µl of rSAP to 5 µl of PCR product. (Note that the Quick Dephosphorylation Kit (NEB #M0508), which contains Quick CIP, can be used in the same volume).

2. Incubate the mix at 37°C for 15 minutes.

3. Inactivate both enzymes at 80°C for 15 minutes.

4. PCR products are ready for downstream application.

Enzymatic cleanup with Exo I and rSAP is a convenient way of conditioning a PCR product for downstream applications or analysis. It combines minimal hands on time with virtually no sample loss, and enables high quality sequencing results.