Sal I cloning problems - (Jan/24/2008 )
I got only a few more colonies in actual ligation vs control but I got my clone when checked them.
Thanks Scolix. I'll try 2 hours. I've screened a couple but not all the colonies (i have to admit). I did two separate transformation and only screened the first lot. I'll do that next week as well. I've actually had a plate before with one colony and bingo, it was in the correct orientation and sequenced beautifully. Funny how it works sometimes.
Cheers, Rob
over a pcr products, you should purify your product and do an overnight digestion with salI only at 37° (i did them on cell incubator so no evaporation at all).
then the morning if the second enzyme is compatible add 0.5 µl of sal I and 1µl of the other one.
This protocol gave me the best consistent results for pcr products.
And don't purify on gel your pcr product, seem sal I don't like this porcedure (at least in my hands)
then the morning if the second enzyme is compatible add 0.5 µl of sal I and 1µl of the other one.
This protocol gave me the best consistent results for pcr products.
And don't purify on gel your pcr product, seem sal I don't like this porcedure (at least in my hands)
Thanks Fred. Yeah, i might do an overnight digestion with Sal I, might as well. The consensus seems to be that the digest is inefficient and needs longer incubation. I'll use more units of enzyme too. I should have some results by the end of the week, I'll let you all know how it goes.
I'm not convinced about what you've said about not running out my digestion, i can't see what effect that would have. Having said that, i usually just cleanup the digest with a gel extraction column any way (don't run it out), so what you've mentioned won't be a problem.
Just an update. I emailed NEB to ask why Sal I has trouble digesting PCR products (as mentioned in FAQ #8 on their website http://www.neb.com/nebecomm/products/faqpr...tR0138.asp#858). Their answer was that residual dNTPs inhibit Sal I activity. So if you're cloning with Sal I, make sure you run out and clean up (or just clean up) the PCR before you digest, don't digest in the PCR mix (as you can with many restriction enzymes). Unfortunately this doesn't solve my problem, i always run out the PCR before digestion.
Seeya, Rob