PCR Cloning

Cloning PCR Products

If you want to clone a fragment of DNA into a plasmid or construct, one of the easiest ways to do this is using PCR. If you have the DNA fragment or gene already in a plasmid, and the restriction sites flanking the fragment are compatible with the new plasmid you want to clone it into, restriction enzyme digestion usually works well.

However, if you want to engineer new restriction sites for the fragment, construct deletions of the fragment, or mutate sites within the DNA, PCR or polymerase chain reaction of DNA fragment is one of the easiest and best ways to clone your fragment using PCR.

Designing Primers for PCR Cloning

Primers for PCR cloning are usually longer because of the need to add restriction sites.

Restriction sites are usually 6 bp in length. If you are not going to subclone the PCR product, you need to add nucleotides 5' of the restriction site

This allows the restriction enzyme to bind to and cut the restriction site to be more efficient.

Spacer Restriction site Primer Sequence (designed by primer program)

3 bp --6bp—15-22 bp

PCR Subcloning

(Subclone means to clone the PCR product into a vector before you insert it into your vector of interest. Advantages of this includes the fact that you never have to PCR your insert again, you will always have it in the fridge, and you can grow up as much as you want using bacteria.)

From our experience, cutting PCR products is usually much less efficient than cutting fragments out of vectors. Thus, cutting PCR products and cloning them into your vector directly may not be that efficient and you may have trouble doing this.

That is way subcloning into a subcloning vector is usually best.

TOPO Vectors

TOPO vectors are great for subcloning, although they can be expensive. They will save you time in the long run, and make cloning PCR products easier and more efficient.

The reason for this is that you can easily digest DNA in a plasmid. It is more efficient for the restriction enzyme as it can easily bind to and digest your piece.

Disadvantages are that you must have efficient and optimized PCR conditions. The presence of many bands in your PCR will make it difficult if not impossible to clone your PCR product of interest.

You can usually subclone your PCR product within about 2 days. Ligation takes only 5 minutes and you can plate your bacterial colonies the day of the ligation. Once you pick colonies the next day and grow them in media, you not only amplify your insert, you also now have a bacterial colony of your subcloned insert which you can freeze with glycerol into the liquid nitrogen for years.

This really helps when you need to make another construct with your insert!