Yes it is possible. If the RE leaves 5’ overhangs you can fill-in with a labelled dNTP in the dNTP mixture and incubating with Klenow in the appropriate buffer. It depends on the method of detection facility you have access to. A Taq polymerase which doesn’t add an extra terminal nucleotide can also be used. You should not add primers in this case.

If the RE leaves 3’ overhangs, it is a bit laborious but doable. Alternatively if you only cut from one end and your fragment is not very big you can separate them on a denaturing urea PAGE or a sequencing gel.

As you see it is worse the remedy than the illness and it would be absurd and nonsense to perform such experiments only to clone a fragment. Instead, I suggest that you follow some suggestions above. What you are doing has been done hundreds of thousands times and it worked for most people. So, check your RE on a plasmid to make sure the enzyme has not been left out the freezer, etc...Also on the enzyme catalogue you should respect the manufacturer instructions relating to the digestion at the end of a DNA fragment, star activity, etc... Some enzymes may need more extra nucleotides upstream their restriction site than others, etc.... Try to purify your fragment on a spin column (Kit) after each digestion. Normally 1 hour should be OK.

To make sure your PCR product is OK, I would follow Arjan’s recommendation. You can sequence first your fragment and then cutting it from a plasmid will circumvent your issue..

How long was your digestion?

The digestin time was 5 h in 37 C and 1u XhoI +1u EcoRI

Hi Mohammad, After a 5 h digestion, and assuming you used a "universal" buffer, and also assuming that your PCR product was cleaned up after electrophoresis and before the digestion, your PCR fragment should be fairly well digested. What I would recommend for your next experiment, is to do the digestion overnight, or for several hours, but as single, sequential digestions. Ligation, as you may know, are very susceptible to contamination. Make sure you clean your PCR digested fragment before doing the ligation. Best regards.

Hi Mario,

thank you

My problem is that when I digested the pcr product firstly with (XhoI + R buffer+overnight+37 C) and after ethanol percipitation when they digested with( EcoRI and EcoRi buffer+for three hours+37 C) for second digest, the digested pcr product band on agarose gel become Somewhat thick after second digest that seems to they have split, before do this work I used of double digest with 2x tungo buffer.

Is there a way to understanding that digested pcr product is correct before cloning? as you know It is impossible to detect from the gel.

I think you need to divide your PCR product in three eliquotes and then perform the digestion separately using single enzyme in each of the two tubes and double digestion in the third one. Then compare the results on the gel that way you would be sure of the results

Yes it is possible. If the RE leaves 5’ overhangs you can fill-in with a labelled dNTP in the dNTP mixture and incubating with Klenow in the appropriate buffer. It depends on the method of detection facility you have access to. A Taq polymerase which doesn’t add an extra terminal nucleotide can also be used. You should not add primers in this case.

If the RE leaves 3’ overhangs, it is a bit laborious but doable. Alternatively if you only cut from one end and your fragment is not very big you can separate them on a denaturing urea PAGE or a sequencing gel.

As you see it is worse the remedy than the illness and it would be absurd and nonsense to perform such experiments only to clone a fragment. Instead, I suggest that you follow some suggestions above. What you are doing has been done hundreds of thousands times and it worked for most people. So, check your RE on a plasmid to make sure the enzyme has not been left out the freezer, etc...Also on the enzyme catalogue you should respect the manufacturer instructions relating to the digestion at the end of a DNA fragment, star activity, etc... Some enzymes may need more extra nucleotides upstream their restriction site than others, etc.... Try to purify your fragment on a spin column (Kit) after each digestion. Normally 1 hour should be OK.

To make sure your PCR product is OK, I would follow Arjan’s recommendation. You can sequence first your fragment and then cutting it from a plasmid will circumvent your issue..

I agree with Arjan.......the quickest way is to ligate into similarly digested vector and sequence clones.

I guess you have problem with undigested PCR product for cloning them. A minor issue would be due to the sequence near to the restriction site. For example: your DNA to be cloned may be start with "GAATTC" (site for EcoRI) instead of some short nucleotides AGTTG"GAATTC". This additional bases (random!) is known to enhance the digetion of RE(may be the enzymes find the site better and bind with high affinity..I dont know). Pleae refer the link suggested by NEB:https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

Good luck!

Hi, you should clone your fragment in to TA vector and then you can see the results of the digestion.

Hi friends

thank you for your help

I want to try Arjan suggestion I think it's Simple and reliable

A PCR product cloning vector,TA or blunt or seamless etc., is always the best way to go when concerned about this question.

As an irrelevant aside, a quick way to answer your original question about restriction digest quality control might be to simply put ligase with your digested/purified fragment for 5-60 min then run it on a gel. If only one enzyme cut you will likely only see one or two bands: unligated DNA fragment and a band of double that size where one one the ends ligated together. This is because if, for example, only EcoRI cut, the EcoRI ends of two fragments would ligate together forming a double-length fragment. If, however, both enzymes cut, you could expect to see a ladder of higher-order multimers because the after the EcoRI end ligated, the other end could then ligate to the XhoI site on another fragment becoming 3 fragments long, and the process can repeat. Note that at any even number of fragments the chain has the option of circularizing with itself instead of ligating to another new fragment, thus stopping the chain.

Hi John,

thank U for your interesting idea but

Are you sure about that if both of enzymes work properly we see a ladder of higher-order multimers on the gel? I think we may see smear on the gel instead of ladder because different types of binding may occur in ligate reaction!

I agree with John. I’ve done that with 3 fragments but with the aim to have a cassette not to test a RE enzyme. Mohammed, you can’t have a smear from the sum of two discrete fragment lengths no matter how many time they combine between them.. It is mathematical

Hi

Dear Abdelhalim,

Can you explain more for me please?

I think when we have large number of fragments with two sticky ends, for example A and B, many combine may occur, such as B-A-A-B/A-B-B-A/B-A-A-B-B-A-B/A-B-B-A-A-B/ and etc.maybe Your purpose is about abundance of combining two fragments instead of Other possibilities, is it correct?

Hi Mohammed,

It doesn’t matter how they do combine. Just give them numbers and sum it up. Let say A= 200 bp and B= 300 bp

B-A-A-B =1000 bp

A-B-B-A= 1000 bp

B-A-A-B-B-A- = 1500 bp

A-B-B-A-A-B/ = 1500 bp.

You will detect products with 400 bp (A-A), 500 bp (A-B) 700 bp (A-A-B) (A-B-A), etc…as single bands not as a smear. Of course, if you keep the ligase for longer time you should expect high molecular weight multimere which you may not be able to resolve on a gel.

Hi Abdelhalim,

thank you for your clear description

best regards.