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BsaI – Worst restriction enzyme ever

June 28, 2013

This is public service announcement. BsaI is the worst restriction enzyme ever. Never use it or any protocol that suggests you use it. I know that includes just about every TALE construction protocol, but really save yourself a bunch of pain and suffering and don’t use BsaI.

If you’re in a lab that is making TALE construction platforms, please just stop using this enzyme. Ask yourself why you insist on publishing protocols using this enzyme when there are better options. Your lab must know this enzyme doesn’t work. You have already caused endless hours of wasted time to graduate students and postdocs around the world. Have a quick read of one of the TALE Google groups and it’s an endless stream of failure caused by this horrible enzyme.

To anyone reviewing a methods paper that uses BsaI, please tell the authors that they must rid the protocol of this enzyme before publication. Seriously, no paper should pass review that uses this enzyme.


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  1. Glen Corson permalink

    wow that’s too bad you’ve had so much trouble with BsaI. I’ve been using it (from NEB) a lot lately and it’s been working fine! Even when doing 3-way ligations, which are lower efficiency anyway, I’m getting great success: plenty of recombinant colonies and all my sequenced clones have correctly spliced/reformed site. BsaI has the great feature of cutting outside its recognition site which really adds flexibility to cloning strategies. Just remember that when are you are combining fragments you must be sure the overhangs match, not just the recognition site itself. Just because you can map a BsaI site on two different molecules does NOT mean they will base-pair! Maybe that is where some users go astray. Good luck

  2. ethanomics permalink

    Wow, I’m really surprised it works for you. I had to use a whole tube of it to cut 5 ug of plasmid. After all the successive purifications I was down to 1 ug by the end. One of the sites had a 4 out of 5 bp match to the Dcm recognition site so I wonder if E. coli is not that accurate with Dcm methylation.

    Here are a couple posts from a TALE Google Group:
    “BsaI is not a long lasting enzyme and will stop cutting properly after ~3 hours. Also, it can degrade quickly, even at -20, regardless of what the expiration date says. We started to have difficulty building the TALeEN pieces until we started making aliquots of the BsaI to freeze back to -80 to ensure proper cutting.”

    “Use regular BsaI. BsaI-HF has a mutation (Y231F) which leads to increased fidelity which is great for reducing star activity. However, the Golden Gate reactions has such short cut times, that you want the wild-type enzyme. I’ve never tried longer cycles but my guess is at some point you don’t gain anything as ligase won’t be active after too many rounds of being at 37C.”

    And there are a lot more posts like that. It’s interesting it works so well for you. I wonder what it is. Perhaps there are some requirements outside the defined recognition site that make a difference? Dcm methylase isn’t that accurate?

    Anyway I’ve switched to BsmBI and BspQI neither of which are great enzymes but they’re working a lot better then BsaI.

    OHSU is totally awesome! You gotta love those views of Mt. Hood and Mt. St. Helens.

  3. Berkeley permalink

    Funny, bsai works much better than bsmbi for us.

  4. Seems to be working for us. And our tube has an expiration of 2013!

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