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Digestion of PCR Products

• Cleavage of PCR Products Directly after Amplification Reaction (see below)
• Cleavage Efficiency Close to the Termini of PCR Fragments

Cleavage of PCR Products Directly After Amplification Reaction

The most convenient option for digestion of PCR-amplified DNA is the addition of a restriction enzyme directly to the reaction tube after completion of PCR. All Fermentas restriction enzymes have been assayed in PCR buffers supplemented with all PCR components. The majority of Fermentas restriction enzymes are active in the Fermentas buffers used for PCR.
However, according to our observations, digestion of PCR products is often inefficient, even though the restriction enzymes are 100% active in the PCR mixture prior to any amplification reactions.
Therefore, we recommend dilution of the PCR product at least 3-fold with 1X recommended restriction enzyme buffer prior to digestion.

Protocol for Digestion of PCR Products

1. Add components in the following order:

   PCR reaction mixture (~0.1-0.5 µg of DNA)	10 µl
   Water, nuclease-free	16-17 µl
   10X recommended buffer	2 µl
   Restriction enzyme (10-20 u)	1-2 µl

2. Mix gently and spin down.
3. Incubate at the optimum temperature for 1-16 hours.

Note

• If the diluted PCR products are incompletely digested or not digested at all, purify the PCR products with the DNA Gel Extraction Kit, then digest the purified DNA.
• For cloning applications, purification of PCR products prior to digestion is highly recommended to remove the active thermophilic DNA polymerase still present in the PCR mixture. DNA polymerases may alter the ends of the cleaved DNA and reduce the ligation yield.
• If a restriction enzyme requires special additives (e.g., SAM), reduce the amount of nuclease-free water appropriately.
• PCR additives such as DMSO or glycerol may affect the cleavage efficiency of restriction enzymes or cause star activity.
• When introducing restriction enzyme sites into primers for subsequent digestion and cloning of a PCR product, refer to the Table “Cleavage efficiency close to the termini of PCR fragments” to define the number of extra bases required for efficient cleavage.

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Updated vasario 01, 2008 09:29