I want to set up a taq man reaction. I want to know that in taq man reaction I have to order HPLC purified Primers or ordinary primers are adequate for me. If you have experience in taq man, please help me in this way.
According to my own experience (over 20 years ago) I had no need to purify the DNA primer on HPLC.
Hi
It is not necessary to use HPLC purified primers. You have to look at the history of oligo synthesis in order to understand why commercial suppliers started suggesting HPLC grade oligos. Usually, the efficiency of oligo synthesis is never 100%. You can have a small fraction of "incomplete" oligos in the mix. For e.g., if you ordered a 24 base oligo, you can have a very small proportion of oligos with 22 or 23 bases. HPLC allows one to obtain pure 24 base oligos. However HPLC purification compromises oligo yield and costs more with no substantial improvement in PCR efficiency
Normally, if your primers are very long then you definitely need some kind of purification method. The actual concentration of your full-length primer is almost always less than 100% (see https://www.idtdna.com/pages/education/decoded/article/getting-enough-full-length-oligo- ). RPC-based HPLC purification should give you around 85% purity.
However, for quantitative PCR such as yours, I don't expect your primers are very long? I normally use primers up to 40 nt in length without any purification, and they work fine.
Good luck!
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