Antarctic Phosphatase treatment - (Nov/20/2010 )

Hi everyone! I just had a question about my restriction digests. I'm digesting a plasmid with RsrII (NEB 4) and EagI (NEB3). I'm digesting w/ RsrII first, then column purifying my digestion. Next, I digest with EagI. And...I was supposed to treat with antarctic phosphatase concurrently, but I forgot. How likely is it that my sticky ends will anneal back together between the time I take the digestion out of the incubator, purify it through the column, and then treat with APase? I don't think this is an extremely common occurrence, but I'm paranoid about my cloning working. Thanks for your input!

That will not be your problem. There may be other issues, but this is not one of them.

RsrII and EagI don't have compatible ends, so why phosphatase treat at all? In any event, phosphatase stops ends from being ligated together -- since there's no ligase involved yet, it doesn't matter when the digestion is treated, so long as it's before the ligation reaction.

We treat with APase after the second cut because our vector contains Amp resistance. If the ends were to anneal together we could possibly get false positives with the ligation. Additionally, although each enzyme has different nick sequences, I'm ligating into the same vector that I'm cutting from...ironic? Yes.

But when the enzymes don't produce compatible ends, they won't be ligated together any more than you'd expect an insert fragment cut with an enzyme that doesn't produce compatible ends with your prepared vector to be successfully ligated...

Phosphatase is usually only used when you are doing a single digest, or a digest which produces compatible ends, and you want to make sure they won't ligate together again. The ends will only ligate together if you have DNA Ligase there, and if they are compatible. As you are cutting with enzymes which produce different sticky ends, then purifying the digest, they won't be able to ligate to each other anyway so the phosphatase isn't required!