I am currently working on preparing a 9 kb sequence of DNA for restriction digestion into the pBAD30 expression vector. There are very few restriction enzymes that do not have a restriction site located on my insert, and since I am using 2 restriction enzymes in my digestion, I had little choice in choosing my restriction enzymes. The only two restriction enzymes that will work for me are Xmal and KpnI. XmaI uses CutSmart buffer while KpnI uses NEB buffer. The efficiency of XmaI in CutSmart buffer is 100% while the efficiency of KpnI in CutSmart buffer is 50%. Would it be easier to perform a double digestion using CutSmart buffer and 2x KpnI than to perform two consecutive digestions?
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1$\begingroup$ Hi Hunter. What conclusion have you come up with yourself? You've received a close vote on your question because another user likely assumes you're looking for others to do your work for you (i.e., figure out which approach is best). I think you'll get a positive response if you can demonstrate a little more explicitly that you've thought about this on your own and that you've spent some time researching to support one way or the other. It will likely sharpen your question and create a more interesting dialogue and answer. Thanks! $\endgroup$– theforestecologist ♦Commented Feb 24, 2019 at 17:58
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1$\begingroup$ I am definately not asking others to work for me. I am simply asking a scientific question that I was unable to find the answer to. I should not have to fear asking a logical scientific question. Closing this question is only holding back the scientific community and preventing others, like me, from learning more about science. $\endgroup$– HLRCommented Feb 24, 2019 at 18:18
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$\begingroup$ Hunter please don't get mad. I'm not even the one that voted to close. I'm just letting you know that some have interpreted your question as asking for an answer to a question you haven't fully indicated you tried answering on your own. If you were to indicate explicitly what you have already done to try to answer the question (e.g., where have you looked, what have you read, etc.), then this concern is avoided. Closing questions might be holding back some individuals, but it's encouraging most to take that extra step toward being effective scientists: self-guided learning. $\endgroup$– theforestecologist ♦Commented Feb 25, 2019 at 0:24
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1$\begingroup$ @theforestecologist I apologize for sounding angry. I will do better in specifying my efforts to solve the problem in the future. Thank you for the advice. $\endgroup$– HLRCommented Feb 25, 2019 at 3:55
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3$\begingroup$ In my opinion question seems to be in correct format (may be I am seeing edited version). @HunterRees question is more of practical than theory.He is facing problem as he is stuck at one of the steps of his experiment.In search of solution, he has explained what he has found (like combination of buffers he mentioned).However, before performing the experiment he is seeking some more advice from the experts on this. $\endgroup$– Science123Commented Feb 25, 2019 at 12:48
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3 Answers
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In my experience, a double digestion in CutSmart buffer will work perfectly well. The reaction may proceed slower, but incubate it a little longer and run a gel after the digestion - you'll see whether it has worked. The other answerers unfortunately did not mention that the best way is to check the restriction products yourself on a gel. Cut out the fragment from the gel you need (and purify) if you want to avoid undigested plasmid. See if the plasmid fragment sizes correspond to the correct cutting sites. Basic lab practice and it takes very little time!
Beware of star activity - ask a technician to sequence your fragments or plasmids to be sure it's all going well (this is good lab practice), in case you see unexpected bands on your gel that may indicate unwanted restriction activity. This is not so common, though :)
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1$\begingroup$ I would have thought checking a restriction digest on a gel goes without saying. Also, is your experience actually using KpnI in CutSmart? NEB explicitly warns that extended digestion with this enzyme may result in star activity, though I don't know how serious of a problem it actually is. $\endgroup$ Commented Feb 25, 2019 at 21:02
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$\begingroup$ Even with star activity, the enzyme is partially doing its job correctly and the plasmid is partially digested correctly. Running the product on the gel and purifying the correct fragment is the best way to do it for three reasons: (i) you know exactly what your product is, (ii) you don't need to purchase, wait and/or troubleshoot new enzymes, and (iii) you're fostering that sweet sweet scientist thinking, rather than being a protocol zombie - my technician's words, not mine :-) There is one quite minor downside: you can't do this with very little quantities of plasmid (does not apply here). $\endgroup$– S PrCommented Feb 26, 2019 at 10:34
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1$\begingroup$ Fair enough. I still prefer to use the optimal buffer, which is especially easy for me since I do sequential digests. To each their own. $\endgroup$ Commented Feb 26, 2019 at 16:49
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NEB double digest planner is suggesting to use 2.1 buffer for your combination of restriction enzymes (XmaI 50% and KpnI 75% activity). I would be more worried about the star activity of KpnI in CutSmart buffer than the lower activity. The latter can be overcomed by prolonging reaction time or using more enzyme.
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If you haven’t already ordered your enzyme, you could get high fidelity KpnI, which has been engineered for use in CutSmart.
You could also consider doing a sequential digest rather than a double digest (ie digest with one enzyme in its optimal buffer then change the buffer for digestion with the second enzyme). It is more work but benefits from easier troubleshooting in case one of your enzymes is not cutting (which can occur frequently if you have a freezer full of 10 year old enzymes).
answered Feb 24, 2019 at 19:03