I am trying to digest a 491bp intl amplified PCR product into various classes but it seems I am not using the right protocol....
@ Doninita, Is there any other better enzymes that can cut like those two for differentiating the classes of integrons? I used White et al protocol
I can provide you sequences of the different classes of integrons such that you can design primers
I have an experience with RsaI - Tango buffer, 37 OC for about 16 hours (overnight).
Dear Dele, it took lots of time to me to understand your problem. Then I figure out that it might be the situation in which you are using the right approach and protocol, but simply you don't have all the integrons there.
For amplification part, you may have a look at http://medind.nic.in/iau/t11/i2/iaut11i2p118.htm
So the right question I should have been asking is which kind of PCR product are you looking into for integrons? I mean what is it. We can talk more after you check the link above, it might be not a problem of amplification nor of digestion...
It may come out that you don't have all integrons present, right?
Donita You are absolutely correct, it seems I actually did not have the integrons present, perhaps the primer i used wasn't good enough. I guess if i had used a primer that will detect all the variable region, I eventually solved the problem.However all your contributions went a long way as well as Thomas and Zornitsa excellent and intellectual contributions were helpful.
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