I have done qualitative PCR for a gene and would like to sequence using Sanger. Should I use the normal PCR or can I use the qPCR products, as I am not sure if the SYBR Green dye will interfere.
Hi Anuradha,
I guess it will depend on the polymerase you want to use for sequencing. I have heard that T4 ligase is inhibited by SYBR green...
You will probably have to clean up your PCR product anyway to get rid of the primers - so why not invest the time and do another conventional PCR yust to be shure?
Hi,
Thank you for the answer, I am thinking of doing that actually, just was curious. I am using BigDye pol for sequencing.
I am interested in the answers to this question, as I would like to do the same thing. Would a standard clean-up work? The SYBR green intercalates with the PCR product, so I wonder whether all clean-up methods would remove it. Maybe it's not a problem if it's still in there anyway
I imagine that using the qPCR product as template for a standard PCR would work, because the new amplicons would not have SYBR green.
Maybe you have tried to sequence the product now. If so, did it work?
http://www.lifescience.net/protocols/52/sybr-green-qpcr-with-standard-curve-protocol/
See step 5. Yes you can, but you need to clean up the reaction first.
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