Any suggestions for direct sequencing from large plasmids (10-16 kb)? It's not working very well.

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Yashwant Chavan

You can also use 5% DMSO in sequencing reaction, this can help in denaturation of plasmid DNA. Further for such plasmid DNA (10-16kb) DNA concentration required for sequencing would be high. As Number of copies of plasmid DNA in 100ng of DNA in would be less in such cases.

Harrie Van Erp

Thanks for your suggestions. I will try heat denaturation. Supercoiling might be a problem.