Double Digestion

Restriction enzyme digestion became a routine method of molecular biology 2 decades ago. Till now researches use restriction enzymes for cloning, analysis of genomic sequences and DNA methylation. Here I give a short overview on the usage of restriction enzymes and some tips from my practical experience.

Original Message
Date:01/07/2013 08:58:00
Thanks a lot for your reply, BlueDevil. I used vector primer, RV3 to sequence the plasmid, and at the same time the other clone had been successfully sequenced. About the plasmid quality problem I guess I would rule out by the second time sequencing because that I sent them the streak strain. If there's any wrong conclusion please correct me. I wonder two things: 1) is it possible that the cloned plasmid passes double digestion test but failed in sequencing? what reasons cause it? 2) will re-transform the plasmid and send the new colony afterward solve the problem?
Thanks a lot.

1) is it possible that the cloned plasmid passes double digestion test but failed in sequencing?
With molecular biology, anything is possible. I'm assuming you are using a selectable marker, e.g. ampicillin resistence. Is that correct? If so, and if we assume the selection agent is at the correct concentration and the plates are not too old, the bacteria must contain plasmid. The fact that you carried out double digestion successfully also rules out the absence of plasmid. Although seeing a band of the expected size (was it?) on a gel after double digestion is very suggestive of the presence of your insert, only sequencing will tell you for sure. If another clone in the same vector was sequenced successfully, it sounds like something is wrong with your plasmid prep. You might try re-isolating your plasmid, and the plasmid containing the insert that was successfully sequenced, and sending them in at the same time. If neither sequence, it's the prep. If only the one that worked previously sequences, then it's the new clone.

2) will re-transform the plasmid and send the new colony afterward solve the problem?
It might, but if the problem is in the prep, it probably won't. How are you purifying the plasmid? Even with a good plasmid purification kit, and depending on the bacterial strain, it sometimes takes some additional clean-up to get things to work. I would suggest an extra ethanol precipitation; make sure you wash the pellet thoroughly with 70-75% ethanol before air drying. Resuspend the plasmid in water and make sure your water is very good. The same goes for the primers. Did you include primers for sequencing from both directions?

Thanks a lot for your reply, BlueDevil. I used vector primer, RV3 to sequence the plasmid, and at the same time the other clone had been successfully sequenced. About the plasmid quality problem I guess I would rule out by the second time sequencing because that I sent them the streak strain. If there's any wrong conclusion please correct me. I wonder two things: 1) is it possible that the cloned plasmid passes double digestion test but failed in sequencing? what reasons cause it? 2) will re-transform the plasmid and send the new colony afterward solve the problem?
Thanks a lot.

Just a couple of questions: What primers are you sending for sequencing? Are they outside the insert or contained within the insert? If they are inside the insert, it's possible the insert is not what you think it is. Is it possible to use primers that are part of the plasmid sequence? Did you send the entire plasmid or just the insert?

Did the company give you any more information? It's possible to have sequencing not work very well if you use too much, or too little DNA, or if the prep is dirty. The company should be able to give you a hint as to what they think the problem is.

Have you sequenced anything else successfully with this same company?

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Hello guys, I have a problem that drives me crazy for a while.
Here's the situation: I finally made clones after ligation of TA-clone-insert into pGL4.17, which has reporter gene for assay. Then I checked by colony-PCR and specific double digestion by KpnI and HindIII, after I saw clear insert band cleaved from the clone, I sent it to do auto-sequencing. The first time I sent the plasmid which I'd checked by double digestion, and the company said there's no signals (they supposed the solution has more than one plasmid), so I decided to streak and sent plate for sequencing. However the second time still reported no signals which the company commented impure strains. All I can think is re-transdorm the plasdmid I checked, and then select one colony to streak for sequencing, but if the two statement from the company is true, then will my re-transformation solve the problem?
Thank you and look forward to your reply.

Thanks for posting this! It is very helpful

Hi,

I'm reading your problem here, and it's very similar to what I've been stuck with for quite a long time. I used BamHI and XbaI cutting sites in one of streptococcal vectors, and somehow the stuff didn't work. If I did separate digests, I would get some sort of partial digestion with XbaI, and all fine with BamHI. Yes I suspected the scrambled sites, even sequenced it - was fine. Sites are 12 bp apart. All lab gone nuts. And guess what was my problem, when we figured it out in the end. dam methylation! Dam(n) methylation, as I say now. XbaI is methylation-sensitive, and my vector was cultivated in E. coli DH5a, which are, sadly, Dam-positive. So obvious when you know, but so easy to overlook. However, partial digestion was possible, I assume it's due to the incomplete methylation during fast replication of plasmids, when you cultivate them for extraction. Some sites remain not perfectly methylated, and XbaI is able to get to that one.
Maybe it's totally not your case, then sorry, but I thought I'd share my really bloody experience. Best luck in your work.

Original Message
From:pEBlue-28
Date:07/08/2012 01:01:00
Thank you Bluedevil.. The size of PCR product is about 560 bp and after double digestion it is about 550bp. Yes The enzymes work well. I have cheked them individualy and they could cut plasmids. I used Poly acrylamide but native one. One strange thing that I faced is that after gel extraction the size was OK! What might be happening?

So, after double digestion the PCR product runs marginally faster than before (560 bp before and 550 bp after). And, the size is fine after extraction from the gel. I don't understand the problem. The PCR product should be smaller after double digestion; the exact amount smaller dependent on how many bps were removed. Of course the ultimate test is sequencing (either before or after ligation into a vector) but, so far, everything seems OK.

Thank you Bluedevil.. The size of PCR product is about 560 bp and after double digestion it is about 550bp. Yes The enzymes work well. I have cheked them individualy and they could cut plasmids. I used Poly acrylamide but native one. One strange thing that I faced is that after gel extraction the size was OK! What might be happening?

What is the size of the original PCR product and what is the size after double digestion? Have you tried using the enzymes individually, followed by an agarose gel, to see if you can narrow the problem down to one or the other of the enzymes? Have you tried a denaturing polyacrylamide gel (link?

Do the enzymes cut a plasmid successfully when used individually?

Hi guys, I face a problem during my cloning project. The PCR product size using pfu Polymerase was OK but when I double digested it I saw that It moves so slow and the size is not matching!. I deactived both enzymes but it seems that the double digested inserts are aggregated or maybe it is because of the influence of Res enzymes on the movement!. Am I right? This problem was happened even during cloning confirmation using double digestion reaction on recombinnat plasmid.

Hi
Thanks for your advise merwyn1978.
BlueDevil, I happily must say finally it worked and I got the band but with the same enzymes and buffers. I don't know what was the exact reason. maybe my vector concentration was not optimum, and maybe anything else.
In any case, time solved it (of course ruined my last 4 months).
My supervisor says whenever you don't get result for long time with no reason, give it up, go home or a trip, then it will be solved.

Very glad to hear it! Thanks for the update.

Hi
Thanks for your advise merwyn1978.
BlueDevil, I happily must say finally it worked and I got the band but with the same enzymes and buffers. I don't know what was the exact reason. maybe my vector concentration was not optimum, and maybe anything else.
In any case, time solved it (of course ruined my last 4 months).
My supervisor says whenever you don't get result for long time with no reason, give it up, go home or a trip, then it will be solved.

Hi
Your problem is simple. Both of your enzymes work, but they are not that much efficient, such that u view them in gel. Go for a bulk 100 microlitre reaction. Use the buffer for the enzyme which takes longer time to cut and add both the enzymes and complete the reaction. Then purify the sample with pcr purifying kit, then run on gel. u can see the work. coz this worked for me.

It would be my pleasure. I will let you know as soon as I find out.
Thanks so much for your attention.

I have changed buffers several times; after extraction of first cut product from gel, I used two kinds of buffer for XbaI. NotI works well with Orange buffer, XbaI do either. For second cut by XbaI, I used both Orange and Green Buffer (remember my XbaI is Fast digest from Fermentas). No change in result. TA-vector has some digestion sites in poly linker region, I used 2 kinds of enzymes which were able to cut 2 of those sites. Since they were from vector, and there was a fragment between them, they should work, but didn't.
I think there is some thing that inhibit my second cut, whether it want to be TA-vector or PET26 or an expression vector. I want to ask a friend to repeat double cutting for me but completely separately in other lab. I hope it work, because I must double cut the mentioned expression vector and use it for my own recombinant gen to express it in CHO cells.

Definitely a tough problem. But, XbaI/NotI digests are very common. I have yet to see a report of one enzyme inhibiting the second, especially with sequential digests. If we assume that your enzymes are working, the only possibility left is the construct. My money is still on the construct. If the construct has one XbaI site and one NotI site, separated by 750 bps, they should both cut. When you figure this thing out, would you mind posting what you find? Good luck!

I have changed buffers several times; after extraction of first cut product from gel, I used two kinds of buffer for XbaI. NotI works well with Orange buffer, XbaI do either. For second cut by XbaI, I used both Orange and Green Buffer (remember my XbaI is Fast digest from Fermentas). No change in result. TA-vector has some digestion sites in poly linker region, I used 2 kinds of enzymes which were able to cut 2 of those sites. Since they were from vector, and there was a fragment between them, they should work, but didn't.
I think there is some thing that inhibit my second cut, whether it want to be TA-vector or PET26 or an expression vector. I want to ask a friend to repeat double cutting for me but completely separately in other lab. I hope it work, because I must double cut the mentioned expression vector and use it for my own recombinant gen to express it in CHO cells.

Just one cut.
For simultaneous digestion (NotI needs 3 hour incubation and XbaI is fast digest and 30 min is enough), I incubate with NotI and after 2.30, I divide it into two parts, one part goes on just with NotI and another part XbaI is added. There are two other microtupes too in which one has only XbaI and another one, uncut.
Bands of three types digestion (two single and one double digestion) are exactly same.

Very weird. Each enzyme cuts individually but does not cut if used after the first enzyme. This is totally bizarre. The only explanation is that, somehow, the first enzyme screws up the site for the second enzyme, no matter what order you do the digests.

With the double digestion, you say you only get a single cut. Do you know which enzyme is cutting? I'm still thinking you may want to sequence relevant parts of the construct after digestion with each enzyme. At least you should get new enzymes and use new buffers.

Just one cut.
For simultaneous digestion (NotI needs 3 hour incubation and XbaI is fast digest and 30 min is enough), I incubate with NotI and after 2.30, I divide it into two parts, one part goes on just with NotI and another part XbaI is added. There are two other microtupes too in which one has only XbaI and another one, uncut.
Bands of three types digestion (two single and one double digestion) are exactly same.