Exonuclease I - Shrimp Alkaline Phosphatase Clean Up of PCR Products

ExoSAP protocol for purifying PCR fragments for automated DNA sequencing using SAP and Exonuclease I

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Overview

The ExoSAP protocol is the simplest way to clean up PCR products before sequencing. The exonuclease I removes leftover primers, while the Shrimp Alkaline Phosphatase removes the dNTPs. This nice thing about this protocol is that you don't need to pipette your PCR products out of their original tubes thus minimizing the potential for PCR contamination of your lab and equipment.

A warning for people in USA, Canada and Japan. ExoSAP-IT is covered by patents owned by Affymetrix, including U.S. Patent Nos. 6,379,940 and 6,387,634 and corresponding patents in Canada and Japan. These products or portions thereof are sold under license from GE Healthcare under US Patent Nos. 5,741,676 and 5,756,285, and corresponding patents issued in other countries. This means that if you are in one of these three countries that you should not use the following protocol, but instead purchase it from Affymetrix.

1. Description of ExoSAP Process

Enzymatic removal of excess nucleotides and primers from PCR reactions.

2. Chemicals involved in EXoSAP Process

3. Potential Hazards

4. Personal Protective Equipment

Wear a lab coat, enclosed shoes, safety glasses or goggles and latex or nitrile gloves for eye and skin protection.

5. Engineering or Ventilation Controls

None.

6. Special Handling Procedures and Storage Requirements

Do not touch the hot plate or lid of the PCR machine when in operation.

7. Spill and Accident Procedures

8. Decontamination Procedures

Wipe up any spills with paper towels and water. Dispose of contaminated waste in the clinical waste container.

9. Waste Disposal Procedures

Disposal of the accumulated waste in the appropriate waste disposal container.

ExoSAP Protocol

This protocol is used for 25µL PCR products that require removal of excess dNTPs and primers prior to sequencing. The final volume of the reaction is 32µl after ExoSAP treatment (accounting for 3µL being run on an agarose gel previously). From this, 3µL of PCR product is normally sufficient for performing a DNA sequencing reaction using BigDye. This protocol can be adapted by reducing the volume of MilliQ water added to the ExoSAP master mix should a smaller PCR volume be used.

Materials

Protocol

1. Prepare a master mix of ExoSAP for use on PCR products, make mix for the required number of number, plus 10% extra. Prepare master mix immediately before use and keep on ice. Only remove enzymes from the freezer immediately prior to use and use in a cold box. Return enzymes to the freezer immediately after taking aliquot.

ExoSAP Mix            1x               85x             100x

Exonuclease I           0.025µL       2.125µL         2.5µL

SAP                         0.250µL       21.25µL          25µL

MilliQ water             9.725µL       826.63µL        972.5µL

Final Volume            10µL          850µL          1000µL

2. Add 10µL of the 1x ExoSAP mix to each PCR sample.

3. Incubate samples at 37˚C for 30 minutes and then 95˚C for 5 minutes in a PCR machine.

4. Store samples at 4˚C or -20˚C until needed.

Hints

It is a good idea to aliquot out the SAP into small lots (20 µL) and store them at -70˚C. We have found that the SAP tends to become inactivated after a few months of storage at -20˚C, especially if it is stored in a frost-free freezer.