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.
1. Description of ExoSAP Process
Enzymatic removal of excess nucleotides and primers from PCR reactions.
2. Chemicals involved in EXoSAP Process
- Tris-HCl
- Magnesium Chloride All chemicals used in the process are
- Triton X 100 in 1-50mM concentrations in 20µL volumes.
- KCl
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
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
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.
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 a sequencing reaction. This protocol can be adapted by reducing the volume of Milli Q added to the ExoSAP master mix, should a smaller PCR volume be required.
Materials
- Shrimp Alkaline Phosphatase (1U/µL) (Roche Cat. No. 1 758 250)
- Exonuclease I ( 20U/µL) (New England Biolabs Cat. No. M0293S)
- PCR products in 0.2mL PCR tubes
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 aliquots.
ExoSAP Mix 1x 85x 100x
Exonuclease I 0.025µL 2.125µL 2.5µL
SAP 0.250µL 21.25µL 25µL
Milli Q 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.