May 16, 2020

Public workspaceEnzymatic PCR Cleanup Protocol (NEB #M0525) V.1

DOI
dx.doi.org/10.17504/protocols.io.7r4hm8w
  • 1New England Biolabs
  • New England Biolabs (NEB)
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DOI: dx.doi.org/10.17504/protocols.io.7r4hm8w
External link: https://neb.com/protocols/2019/06/04/enzymatic-pcr-cleanup-protocol-neb-m0525
Protocol Citation: New England Biolabs 2020. Enzymatic PCR Cleanup Protocol (NEB #M0525). protocols.io https://dx.doi.org/10.17504/protocols.io.7r4hm8w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 27, 2019
Last Modified: March 29, 2021
Protocol Integer ID: 28188
Keywords: exo sap, exo-sap, exosap, exocip, exo-cip, exo-cip, PCR Cleanup, PCR Clean up
Abstract

Enzymatic PCR Cleanup Protocol (NEB #M0525) uses Quick CIP; Quick CIP is a heat-labile version of calf intestinal alkaline phosphatase (CIP) purified from a recombinant source.


Features

  • Rapid and irreversible heat inactivation eliminates unwanted activity
  • Improved storage stability versus native enzyme
  • Faster reaction setup (no supplemental additives like zinc required) and shorter incubation time
  • Flexible reaction conditions (active in any restriction enzyme buffer, no clean-up required)
  • Less enzyme required (high specific activity), resulting in a lower cost per reaction
  • No need for multiple phosphatases (Quick CIP removes 5′- and 3′- phosphates from DNA, RNA and dNTPs )
  • Active on unincorporated dNTPs in PCR products - improves DNA sequencing and SNP analysis
  • Recombinant for purity, consistency and value
Materials
MATERIALS
ReagentQuick CIP New England BiolabsCatalog #M0525
ReagentExonuclease I (E. coli)New England BiolabsCatalog #M0293
ReagentShrimp Alkaline Phosphatase (rSAP)New England BiolabsCatalog #M0371
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Add Amount1 µL of Quick CIP (NEB #M0525) to Amount5 µL of PCR product.
Note
Note that 0.5 μl of Exo I (NEB #M0293) and 1 μl of rSAP (NEB #M0371) can be used in the same volume.


Pipetting
Incubate the mix at the Temperature37 °C for Duration00:15:00 .

Incubation
Inactivate both enzymes at Temperature80 °C for Duration00:15:00 .

PCR products are ready for downstream application.