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The error rate of PCR is still very high in comparison to natural bacteria DNA replication of its plasmids. Natural bacteria DNA replication has an error rate of approximately 1 in 10 billion, and the best PCR polymerase commercially available (Q5 from NEB) has an error rate of approximately 1 in 1 million. Therefore, cloning fragments into bacteria and ...


You can use PCR products in Sanger sequencing; it is very common. Using PCR products instead of cloned genes does raise a set of problems that are less of a concern than with cloned sequences, such as the presence of incomplete or incorrect PCR products, but there are standard and simple solutions for most of these concerns.


From my experience I would say that it is no problem to freeze the digested DNA, when the enzyme is inactivated (even when not, for this time period I wouldn't expect much damage). However, if possible, I would rather opt to freeze the PCR product and make the digest fresh, since the overhanging ends are kind of sensitive to hydrolytic degradation. I have ...

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