Jul 18, 2023
20 minute PCR Enzymatic Cleanup
- Julian Liber1
- 1Department of Biology - Duke University
Protocol Citation: Julian Liber 2023. 20 minute PCR Enzymatic Cleanup. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1q4bkgr2/v1
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: July 17, 2023
Last Modified: July 18, 2023
Protocol Integer ID: 85121
Keywords: minute pcr enzymatic cleanup, pcr product for cleanup, enzymatic cleanup, sanger sequencing, pcr product, compatible protocol for cleanup, pcr, minute pcr, sequencing reaction, thermocycler, downstream sanger, primer, cleanup, calf intestinal phosphatase, ssdna, intestinal phosphatase
Abstract
A rapid microplate-compatible protocol for cleanup of PCR products prior to Sanger sequencing. Verify that the PCR produces a single band, and retain some PCR product for cleanup and sequencing. Set up at room temperature, and react on the thermocycler.
Mechanism: PCR products contain 1) primers and 2) dNTPs which will interfere with downstream Sanger sequencing reactions. Exonuclease I degrades ssDNA (primers) and calf intestinal phosphatase degrades dNTPs. Functions like NEB #E1050.
Materials
- Quick CIP (5U/uL) NEB #M0525
- Thermolabile Exonuclease I (20U/uL) NEB #M0568
- PCR-grade water
Troubleshooting
Making the enzyme master mix
Add to a 1.5 mL sterile conical tube
- 760 uL water
- 94 uL 10x rCutSmart buffer
- 96 uL 10x Exonuclease I buffer (NEBuffer r3.1)
- 4 uL Quick CIP (5U/uL) NEB #M0525
- 10 uL Thermolabile Exonuclease I (20U/uL) NEB #M0568
Mix, then aliquot 200 uL into separate tubes. Store at -20C. Good for at least 2 years.
PCR Cleanup
Mix 1.2 uL of master mix with 1.5 uL PCR product in PCR strip tubes or plates.
Note
A 1:1 ratio of enzyme mix:PCR product can also be used. Pipetting 2 uL may be easier, and you can do equal parts in such a case. If doing a large number of reactions, I generally use 2 uL because it is faster to pipette that volume and ensure it stays in the tube.
Seal tubes or plates and place on a thermocycler. React at 37ºC for 15 minutes, then 80ºC for 1 minute.
Add the product to Sanger sequencing reactions. 1 uL of product, 1 uL of 10 uM primer, and 10 uL of water per tube. If the band is faint, you can use 2 uL or more of product.
Note
Follow the sequencing facility's requirements for template DNA and primer concentration. The above amounts are recommendations based on what has worked for us.