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The truth is not always what it appears to be


Standard Vector with restriction sites

ItsaMyth

The fact is:

The standard vectors from Eurofins have been designed to have few restriction enzymes in the multiple cloning site (MCS). Even if your restriction enzyme is present in the MCS this will not cause a problem. The extra fragment will be so small that it will not be visible on an agarose gel. All restriction sites in the MCS are at least 4bp (in most cases >10bp) away from your gene so the efficiency of the restriction digest will also not be affected.

In the very unlikely event that one of your chosen restriction sites is also in the vector backbone and a band the same or similar length as your gene is present after the digest, there are a number of possibilities to get around this problem.

Either a 3rd enzyme can be used in the digest that cuts in the vector backbone reducing the size of the unwanted band. In the majority of cases the required band can then be easily gel eluted from an agarose gel.

If this strategy does not work due to the lack of an appropriate restriction site then the gene can be amplified with PCR primers. It is then possible to digest the PCR product with the required enzymes and proceed with the subcloning. It is important in this case to add 2-3 bases to the 5´ ends of the PCR primers as overhangs so the efficiency of the digest is not reduced.

 

 

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