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LH PCR gives wide range of ampilcons

suyiko
in Genetics

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Hello,

 

I am using E8F and E533R primers to amplify 16S rRNA genes from soil bacteria samples and observed a broad range of amplicons from 63 to 663bp.

 

Using the same set of primers and PCR conditions, the same set of samples were amplified and ligated for cloning and yet the clone inserts ranged from 113 to 800bp.

 

Does anyone have similar experiences?

 

What do you think this difference in range is due to?

 

Isn't these set of primers supposed to prime only about 500bp of the 16S rRNA gene? Why is the range much shorter and longer than expected?

 

Looking forward to hearing from your experiences.

 

Thank you.

Generally in these cases the running conditions are not stringent enough (e.g. buffer, too low annealing temp, too many cycles etc.). Or there are DNA contaminations.

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