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digestion problems - (Apr/14/2005 )

dear all,

i am trying to digest my vector with NcoI and SalI , in order to clone another piece in it, also cut with those same enzymes.

when i digest with both enzymes at once, i get 3 bands, although when i check the sequence of the vector it only contains 1 site for each respective enzyme.
when i digest separately with each one, i get one band corresponding to the linearized vector.
if i digest with one and *then* the second, i get one band after the first digestion and 3 after the second.

what could be causing this? any ideas?

thanks a lot!



This is certainly a strange one, and quite frankly it doesn't make alot of sense as I'm sure you will agree.

Let me get this straight if you cut with Sal1 first (1 band right size for vector) and then Nco1 (3 bands - do the sizes add up to the vector?). If you cut with Nco1 first (1 band - vector size) and Sal1 second (3 bands).

The only thing that I can think of is that the enzyme concentration or buffer composition of combining the two results in Star activity.

When you digest sequentially do you clean up after digestion? If not I'd suggest giving that a go to get rid of the previous enzyme and change buffers. Also if possible I'd try another batch of enzyme, buffer and BSA. Other than that I'm as stumped as you.