Problem with DNA Sequencing of qPCR

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Carol12
Carol12's picture
Problem with DNA Sequencing of qPCR

 Hi everyone,

I need help!!

I did a qPCR for a bacteria in lung samples. After that, I sent some samples for DNA sequencing. Just few of my samples are the bacteria I'm looking for, most of them are uncultered bacterium - my bacteria is uncultered as well.

Why is it happening? Poor primer design? Any suggestions?

Thanks
Carol

hankm394
hankm394's picture
Hi,

Hi,
Yes, it is possible that the primers may not have been designed accurately. I would suggest you to evaluate your primers using NetPrimer, which is freely accessible.
Here is the link to NetPrimer:
http://www.premierbiosoft.com/netprimer/index.htm
Also, I found a great resource that claims to be exclusively designed for bacterial identification:
http://www.premierbiosoft.com/bacterial-identification/realtime-PCR/species-identification.html

Hope this helps.
Hank