Recently, I took some deep sea samples and extracted DNA from the sample. I want to know the abundance of the community (bacteria and archaea). So I tried to do the qPCR. When I search the articles, there are so many articles which use different sets of primer.
I have a question that how do I choose an optimal primers to detect my sample? Is there some rules for choosing primers?
Basically in PCR primer designing is must. You may use a variety of software to designs primer for specific genes or segment of DNA. These software will help you to eradicate those primers which causes the formation of cross homology or secondary structure and increases the specificity towards your gene of interest. I am giving a link from which u can download soft wares. best of luck
http://www.premierbiosoft.com/crm/jsp/com/pbi/crm/clientside/ProductList.jsp
Thank you, Mohammad Shawan. I had installed the software. My question is how to evaluate these primes and choose an optimal ones.
Thank you very much, Joshua Mackie! I will consider your suggestions. I think some advice are nice. I have read the article you mentioned evaluating primers. But I think these primers may not good for qPCR, the amplicon(400bp) is a bit long for SYBR Green method. Usually, many articles use the amplicon is about 100-200bp for SYBR Green.
I use absolute quantity PCR method. In your answer you mentioned relative quantity. I want to know more in details. Do you add two set of primers in qPCR reaction.
As you say, extraction method should be improve and avoid variation.
thank you for your help!
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