I am starting to design my primers for cloning. In one protocol, it says that avoid a T as ultimate base at the 3' end. I wonder what will happen if I use T as ultimate base at the 3' end. Is it a big problem for all primers or depend on the primer? Thanks!
Hi Xinji,
There are many guidelines that assist in primer design. However you will find that sometimes when you follow all the rules, the primer doesn't work well it is not specific. Sometimes when you follow none they work just fine. The G or C at the end rule is based on the idea that G or C have three hydrogen bonds and A or T have only two. Thus the primer will anneal with more a more stable energy if a G or C or multiple G or C at the front and back of the target. So, it's better to use a G or C, but often an A or T will work just fine. Sometimes when I am in doubt I will design multiple primers so that if one doesn't work I do not have to reorder new ones. Try them at different annealing temperatures and see if they give good and specific amplification. The A and T on the ends will have a lower melting temperature than with a C or G.
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