im looking to isolate and clone a gene into a vector, however my gene contains no restriction sites. My question is, should my pcr primers flank my open reading frame completely and should i add the restriction enzymes i want to use into the primer?
i will suggest that you to introduce appropriate restriction enzymes to the primer if your pcr kits does not contain the restriction enzyme already.
Yes, your primer pair must contain restriction sites of corresponding selected enzymes sites in the vector. This is better that your gene does not contain any restriction sites whihc maybe present in the vector. However, your vector should have multiple cloning sites (MCS). In your case, select a suitable restriction site/s in the vector and add corresponding sequence region of restriction site/s in the primers as flanking regions.
If your vector contains a blunt end restriction site at MCS, you do not need to add any restriction site in the primers, just make sure PCR amplification does not results in ployA overhangs.Restrict your vector with selected enzymes and you can easily clone PCR product of your gene using T4 Ligase cloning or if you are using Infusion cloning, cloning can be done by infusion reaction.
I hope this answer may be helpful for you.
Buy a modern cloning kit like the TOPO TA cloning kit and you can clone in ANY PCR product. No need to do a restriction digest. You can then move your insert into another vector. https://www.thermofisher.com/us/en/home/life-science/cloning/topo/topo-ta-cloning.html
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