sequencing through insertions sequences - (Apr/15/2006 )

I am sequencing plasmids that I have isolated from faecal material. As a start, I am simply walking to determine if the plasmid is worth having sequenced by a third party (shot-gun cloning with 5X-8X coverage). The problem is plasmids have multiple copies of insertion sequences (transposons such as IS26), so primers that are designed for these sequences gives a dirty sequence. What I have tried to do is design a primer just before the IS in the hope that I read through it so I can design the next primer after the IS. This has worked sometimes but not all the time. Does anyone have a nifty idea on how to get around it? Perhaps a PCR-based method using an IS primers and a random primer?..I don't know..anything! Thanks.

Do just what they're going to do if you send it off for multiple coverage sequencing -- shotgun clone fragments of your plasmid, and sequence the inserts using vector borne primers?

You could try Panhandle PCR. See this thread for more infomation http://www.protocol-online.org/forums/inde...pic=10815&st=15

Daniel

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