I have digested my PCR product with NEB Not1 and Nde1 for 1hr. I have cut the band and purified the product. However I afraid the digestion is incomplete, can I re-digest them again? Is there any side effect other than the loss of sample?
Dear Yen,
That is rightly anticipated. By principle you can digest your samples again, but you will suffer a significant loss in quantity. A piece of advice - set up restriction digests overnight or at least longer than an hour(has always worked for me).
Abhishek
In my experience, you can do it and be effective but you need some good skills:
- purify carefully with phenol/chloroform your digestion
digest again
you can lose sample of course.....but if you do the phenol purification properly, you do not lose that much, and of course you purify your sample again with phenol because if it is done properly is an ancient way that does not waste that much sample
to do a proper phenol purification you need very high quality and "fresh" not oxided phenol/chlorofom solution and be a good in that
it is worth especially if your attempting to clone your sample, so even if you lose sample you will replicate the sample as far as you get enough to transform it....it may be wise to clone in coli so you will easily raise a lot of new sample if needed
good luck :)
You may have thought of this, but one reason directly cloning a PCR product into a vector may not work is if the restriction sites are too close to the end of the product. The NEB catalogue will tell you how well different enzymes work near the ends of DNA. When you design your primers, it's always best to add some extra sequence between the 5' end and the site you will use. This can also be an issue if you are cutting the vector at two sites that are very close. As far as redigesting goes, you can always do it and just see how well the ligation works the next day. The only rule for subcloning is that you confirm that the plasmid you are trying to make is correct at the end.
Hi Yen,
Can I ask what makes you believe that your digestion was incomplete? Normally, 1 unit of enzyme can cut ~1ug of DNA in 15 minutes. So for most standard RE digestions, 1 hour is sufficient to cut the majority of sites needed for cloning. Did you use compatible buffers? NEB 3.1 I think is what is required for those 2 RE's.
Hi Yen,
I assume you think the digestion is incomplete because the cloning didn't work afterwards, right? So just some tips for cloning PCR products after digestion. As mentioned by Michael, it is usually very important to add extra nucleotides at the 3' end of your primers (3' from the enzyme cutting site) adding 6 random extra nucleotides should do for any enzyme. I do it regularly, irrespective of the restriction enzyme I use, since it doesn't harm and then I am in the safe side. In my experience, using a 10-fold digestion respect to the recommended enzyme amount/time is the best way to ensure an almost complete digestion. Meaning that, with a regular enzyme (not FastDigest), I use ten units of the enzyme per microgram of target DNA, 50 microL final volume, one hour, or alternatively use 0.1 U for ten hours or 0.05 U for 20 hours, and so on. One exception for this rule are the enzymes that specifically warn you about the star activity in the datasheet, in this case I attach to the rule of 1U/1 DNA microgram per hour and hope for the best. Another important thing to consider is how you are cutting your band out of the gel. UV can really harm sticky ends, and DNA in general,so I always try to expose the samples the minimal possible time, by just quickly marking the square I want to cut in the gel and turning off the UV light. It is also very recommendable using some thick plastic between your gel and the UV light source, to expose the DNA to less UV. The UV thing caused me problems some years ago with a cloning, so it really can happen. One trick to avoid the cutting all together is loading a small aliquot of your PCR product in a gel and, if the band is clean and specific, just clean up your PCR and digest the product, without further cutting out from the gel. I think that by following that tips, you should be able to clone without further problems. Of course, the bigger the insert size is, the harder to clone. Small inserts(less than 1 kb) should work fine in any range of insert:vector ratio (from 1:1 to 5:1). Bigger inserts could be tricky, I would try a range of insert:vector ratio (from 1:1 to 10:1). Also, for really big inserts(3Kb or bigger), overnight incubation of the ligation mix at 4º has proven to be useful, at least in my hands. Also, in your specific case, NdeI leaves only two overhanging nucleotides, so the ligation can be a little bit more problematic.
And, to directly answer your question, yes, you can re-digest your product, there should be no problem. Depending on the concentration, you may need to phenol-chloroform extract your sample and precipitate , or perhaps you can try to just clean it up with a commercial kit (assuming you have not done that already after digestion).
An remember, you only need one positive clone.
Good luck!
To add some advices to what has been mentioned, you need to verify that the enzymes you are using haven't a star activity if left longer than a hour an dthis information you have it I think in NEB catalog.
Good a Luck
Star activity may be a concern worth bearing in mind if you are using certain traditional, non-modified restriction enzymes. As Abderrahmane suggests, check the manufacturer's specifications, but note that some REs can also start to cut non-specifically after extended times or at high concentrations.
You can redigest your PCR products assume that you recognition site is not at the end of your oligos as answers above. Your should worry about the incomplete digest from your design of the oligos, i.e where your restriction enzymes located.
A lot of restriction enzymes such as Not I and Nde I have low cutting efficiency if the recognition site is right at the end of a short sequence. I assume that your PCR product is at least 150bp long.
Good luck!
Dear Yen Teng Tai,
Yes you can. if any recognition site available then it will cut the rest undigested pcr product. Keep consideration on incubation temperature, sometimes incomplete digestion can figured due to variation in incubation temperature for a longer time.
Wish for the best !:-)
Best regards,
nahid Islam
Yep, just be careful that extended or repetitive digestion can lead to non-specific site digestion
yes but confirm any restriction enzyme have star activity, based on that select incubation time for digestion, this can avoid non specific site digestion.
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