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 any remaining dNTPs. This nice thing about this protocol is that you don’t need to pipette your PCR products out of their original PCR tubes minimizing the potential for PCR contamination of your lab and equipment.
A warning for people located 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 ExoSAP from Affymetrix.
1. Description of ExoSAP Process
Enzymatic removal of excess nucleotides and primers from PCR reactions.
2. Chemicals involved in ExoSAP Process
- Magnesium Chloride
- Triton X 100
3. Potential Hazards
- Low hazard risk from chemicals as they are in relatively low concentrations and microlitre volumes.
- Dry burns from inappropriate operation of PCR machine.
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
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
- Skin Exposure: rinse the affected skin with plenty of water while removing contaminated clothes and shoes. Rinse for at least 15 min. Seek medical attention if needed.
- Eye exposure: Wash eyes for at least 15min, lifting the upper and lower eyelids. Seek medical attention if needed.
- Dry Burn: rinse the affected skin with plenty of water. Rinse for at least 15 min. Seek medical attention if needed.
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.
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.
- rAPid Alkaline Phosphatase (1U/µL) (Sigma-Aldrich Cat. No. 4898133001)
- Exonuclease I ( 20U/µL) (New England Biolabs Cat. No. M0293S)
- PCR products in 0.2mL PCR tubes
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.
|Shrimp Alkaline Phosphatase||0.250µL||21.2µL||25µL|
2. Add 10µL of the 1x ExoSAP mix to each PCR sample.
3. Put the tubes/tray in the PCR machine and incubate samples at 37˚C for 30 minutes and then 95˚C for 5 minutes.
4. Store template samples at 4˚C or -20˚C until needed.
It is a good idea to aliquot out theShrimp Alkaline Phosphatase 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.