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[gandhi]

Hi everyone,

I am new to the forum and this is my first post.  I've never done this before, but I thought it's worth a shot!

I am designing primers to construct a deletion cassette from Pseudomonas aeruginosa and am wondering what is the recommended amount of bp to leave out?

I have normally left out ~300 bp and it has worked fine in Lb. casei under a different system.  However, I am now wondering if I have constructed cassettes that will not recombine with the chromosome due to the amount of bp left out (larger than 1000bp).  I have constructed my cassettes by amplifying 600-800bp of the 5' and 3' regions of the gene, while leaving out critical sights in the middle. There is a selectable marker inbetween my two inserts.

Any help would be much appreciated!

-gandhi