Probably, following notes will be useful:

1. For better hydrolysis all endonucleases should be used with an optimal reaction buffer. As far as I know, both aforementioned enzymes retain 100% of activity only in CutSmart buffer. Therefore, this buffer should be used for double digestion, for other buffers better results will be obtained with sequential digestion. It is crucial to check efficacy of each enzyme by single digestion of plasmid in the same reaction buffer.

2. Insert to vector ratio. Typically, triple excess of insert would be sufficient to achieve preferential ligation of vector with insert. Lenght of insert and vector should be taken in account during calculation of excess.

3. Results in cloning plate can give a hint why cloning is unsuccessful. If there are no colonies, it could be a result of low amount of vector, or over excess of insert taken for ligation reaction. No insert in screened colonies can be a consequence of insufficient hydrolysys or low amount of insert in ligation reaction (beliving that screeening itself works properly). Lastly, it is better to screen around 15-20 colonies, then absence of vector with insert can be proved.

Hope, those observations will help you to overcome problem with cloning.

Hi

You better try overnight ligation at 16°C-22°C and always recommended to check your digestion of your vector by having controls: first tube digetion with only enzyme then second tube with the other enzyme and the third tube digestion with both enzymes (this will help you to assured that both enzymes worked well).

I worked with the same size of insert without troubles.

Goodluck

Hi Yousr,

In addition to the above recommendations, I will like to mention that some restriction enzymes are known to have a low ligation efficiency such as NdeI and require a longer incubation at 16 degrees in order to achieve ligation. It might be good to do a background check on your restriction enzymes.

Secondly, if possible, try to electroporate the ligation mix. It might give you colonies. I had such an experience at some point. You can desalt using specialized filters....I could give you a quote if you are interested.

All the best!

Hello @Yousr,

The above comments are useful and give them a shot, especially if you have time.

I would like to point out two things:

1. some might say that gel purification destroys the ends of an insert. Try to use a commercial PCR clean-up kit and see if there is any difference in the ligation efficiency.

2. Your insert has special ends, but consider using pcDNA 3.1 directional TOPO expression kit. For it, you need blunt-end insert, if that can be done in your case.

Best of luck!

Mirjana

Dear Yousr,

I agree with most of previous recommendations, but I will further recommend to clone the PCR insert without digestion into a intermediate vector as pGEMT, or any other TA cloning if you are using a standard PCR polymerase, and into any other appropriate vector for blunt end fragments if you are using a proofreading polymerase (Spark I or II, for example). After cloning your undigested PCR band it should be easy to release the full double digested fragment and clone it into the double digested vectors (by your results it seems that there is no problem digesting the vector, but insert digestion). Some enzymes do no cut well near the ends of a linear DNA, therefore the only way to digest is after cloning the PCR band into a vector.

Moreover, I agree that ligation will be more efficient at 16 ºC, and in the need of using CutSmart buffer, but 1h should be more than enough with the save time enzymes from NEB.

I hope this could be of some help

Josefa

Agreeing with the previous answers, I would recommend to use competent cells with very high competency purchased from a good company, do not use your own made cells, they are not good enough.

Dear Yousr,

I feel you should keep digestion of PCR products for longer time (8-10 hrs) (for being sure of complete restriction digestion) and the competent cells being used don't have good efficiency. Ligations can be kept O/N at 16-18 degree C. Take 200-300ng of vector and 150-250ng of insert for ligation (in your case).

Good luck.

The above comments are good enough.But if still insufficient, transform all ligation products to competent cell and plate them all.

I would like to add few points,

1. you did not mention about the type of the vector i.e. whether it is a TA vector or Binary or whatever. What I can suggest you is, as the vector size is little higher, try to purify the vector backbone by Electro-elution method instead of normal column purification or something else. It will give a more purified vector backbone where salts and other artifacts will not be present at all.

2. And in case of the amplicons, while running the double digested product on gel, load the amplified product as well as a control and try to run for long time on a bigger length gel so that you can be more sure about the digestion based on mobility shift. Because mobility shift is a better way to confirm the digestion of PCR products than the DNA marker

3. The NEB T4 DNA Ligase works absolutely well if you incubate on ice or 4C for 18-20 hours (that is what we do in our lab). Transform the entire reaction volume, e.g. if the reaction is 15 microliters, transform in 100-200 microliters of competent cells.

Hopefully, it will work out. If still not works, check your PCR product by sequencing once.

Best wishes

Try more modern cloning methods such as Gibson assembly or PIPE. I haven't used traditional double RE cloning since switching to Gibson assembly.

HELLO. Lot has already been said. Would like to add a point here. There are two important points; whether ligation of insert/vector has happened or not? And whether competent cells/strain are efficient enought for such activity?

Confirm your ligated product by using it as template in a PCR reaction using one primer from vector backbone and one from insert. Once you are done with it, focus transformation further. If transformation in e.coli does not work (after changing competent cells/or suitable strain), try to electroporate your ligated product in Agrobacterium cells ( LBA4404, EHA 105 etc). Once it gets stable in it, re-transform to e.coli cells. It works, we confirmed. Good luck

Hi. All the comments above are great but if I can suggest another alternative, I would go with the (often-underestimated) method called AQUA Cloning (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137652).

Basically it's an enzyme-free Gibson assembly method and it works really well. I used it for both small (100bp) and big (5kb) inserts and never had a problem with it. Only time it didn't work well was because of my backbone, for some reason.

Just order a set of primers to open the vector where you want to perform the insertion and a set of primers to amplify your insert on which you add tails that are homologous to your linearized vector (you can go as low as 12bp but 24bp generally works well).

I usually digest my pcr products with dpnI to remove the circular plasmids and then purify on QIAGEN columns but gel purification should work well.

Next I mix my insert and my backbone in a 3:1 molar ratio fashion and let them sit on the bench for 1h at room temp. Finally I transform 25uL of DH5a with 5uL of assembly mix (I usually prepare 10uL) by Heat-shock and plate them on selective plates.

I rarely have a lot of colonies to screen but I often have my clones in it so it's rather quick to verify by colony PCR.

If you need more infos don't hesitate to hit me up! Good luck.

Hi,

There is an other possibility:

" Restriction-free (RF) cloning provides a simple, universal method to precisely insert a DNA fragment into any desired location within a circular plasmid, independent of restriction sites, ligation, or alterations in either the vector or the gene of interest. The technique uses a PCR fragment encoding a gene of interest as a pair of primers in a linear amplification reaction around a circular plasmid. In contrast to QuickChange™ site-directed mutagenesis, which introduces single mutations or small insertions/deletions, RF cloning inserts complete genes without the introduction of unwanted extra residues. The absence of any alterations to the protein as well as the simplicity of both the primer design and the procedure itself makes it suitable for high-throughput expression and ideal for structural genomics. "

Article van den Ent F, L??we JRF cloning: a restriction-free method ...

http://rf-cloning.org/

Hello, I'd like to add another point to all the useful comments: in my experience the gel-purification step (and exposure to UV light) can dramatically decrease the overall cloning efficiency if done with fragments containing single-stranded ends. Therefore a very simple strategy to try to increase the efficiency of your cloning protocol is to skip the agar gel purification step of your target amplicon after the digestion. Given that your protections flanking the restriction sites are 6bp long you can easily get rid of the small DNA fragments (they should be approx. 10bp long) generated after the digestion by a simple round of column purification, therefore retaining only the digested target amplicon, ready for cloning. If possible you should apply a similar strategy to your backbone or load it on a crystal violet agar gel to avoid UV. Fingers crossed.

Hello,

in principal RF cloning, suggested by Karola Almási, is a very good method to avoid restriction problems. In your case I wouldn't suggest this method since the insert fragment is very large. If the advice of Rocco Piazza doesn't work I would try to dephosphorylate the vector before ligating it with the insert. Sometimes this helps preventing religation of the vector without insert in it.

Good luck.

Hi Yousr Dhaou, maybe one approach could be to sequence your insert, to be sure that your restrictions sites are correct. With that, I would try with different plasmid insert ratios, I don't remember if you mentioned the plasmid size. Try with 1:1; 1:2 and 1:3, and transform all your ligation reaction, if possible, avoid gel purification, since more steps will damage the 5' and 3' ends, check if NEB buffer can be used directly in your ligation. Best!!

Thank you all for the great feedback! I have been reading the comments and attempting to implement different techniques with every cloning trial until I got one that works.

What I came to realize is that my cloning has been successful this whole time. My restriction works on my insert and vector, my ligation works and I see this on my gel, and so does my transformation. What I did differently last week to get my clone to show up on my plate was to change the condition under which some genes in my clone will be expressed in the incubation step post-heat-shock. For my system specifically, this is crucial as some genes allow for a less toxic environment and therefore an increased likelihood of surviving cells for colony formation.

I think this is a great option for others to consider when cloning genes that exhibit toxicity and stress when expressed in a host. Again, I experimented with 3 competent cells all of which were highly efficient during previous cloning by other members and this was the sole method that worked.

I've confirmed my cloning with colony PCR, restriction and sequencing.

Thanks for the great thread everyone!

Hi Yousr,

May be you have already resolved the problematic cloning but I would strongly recommend that you use In-Fusion cloning. It is working great.

Regards,

Yordan