pwzl sequencing - (Jun/03/2006 )

Hi evryone,
I'm using a vector pwzl hygro containing the adenoviral protein e1a (750bp) to transfect several human cell lines.
Unluckily there seems to be no e1a expression in hygro-resistant cells, so I need to sequence the construct (I've already did a post-digestion gel, so I know there is an insert there). I did some web search, but I wasn't able to find appropriate sequencing primers nor the pwzl sequence.
Anyone can help?

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First hit on Google:
http://www.addgene.org/pgvec1?f=c&identifi...0674&cmd=findpl

In general, if you don't know anything, you can try M13-F and M13-R, then T7 or SP6. One of these will probably work for most cloning plasmids.

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I found that primer on addgene too, but unluckily there's no 3' primer...

Thanks for the hint about the universal sequencing primers, I'll try them.

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Well, here is a complete sequence for a pWzL plasmid containing an insert. You'll have to dig a little, and figure out where your plasmid insert lies in this structure, but there is enough information to figure that out. The Addgene site shows 31 bp of downstream sequence prior to the insertion, and you know what primer they used, so it should be easy to figure out where the insertion site is located. From there, it should be easy to construct a 3' primer. Good luck.
http://www.stanford.edu/group/blau/maps/b-...-omegaHygro.rtf

You could make life easier for the next person by creating a well annotated plasmid map and putting it on the web.

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