First major suspect is the ATP in your ligation buffer, which progressively hydrolyzes over time. Every time I've compared commercially available ligase buffers to freshly home-made buffers, the latter outperform the former by a considerable margin. Add fresh ATP to your buffer or keep a separate stock and add it directly to your reactions; I use a final conc. of 1 mM. Also, keep a digested plasmid which you know ligates well as a ligase control. A well prepared, singly digested plasmid should yield at least a 100-fold difference between -/+ T4 DNA ligase parallel reactions; less than that indicates inefficient ligation.

Second suspect are your competent cells. Do you have any idea of their transformation frequency? Keep aliquoted portions of some common plasmid (I use pUC19) and include a control transformation using 1 ng in every experiment; transformation efficiency should be at least 10e7/ug (although in real life, routine cloning works perfectly with cells in the 10e6/ug range, provided you add enough DNA).

I hope you are not trying to ligate the shRNA itself in the vector! RNA and DNA make not so good friends ;-)

Now, assuming that you're using the corresponding DNA sequence (and the problem isn't the ATP in the buffer, which as Alejandro said is easy to solve): How did you get it? If you've amplified these by PCR, they'll have blunt ends, which won't ligate with your sticky ends-vector.

It normally works best if you blunt-ligate it first into a plasmid like pJET (don't forget to introduce the desired restriction sites), followed by bacteria transformation, miniprep, double digestion, fragment isolation by electrophoresis and gel extraction, and now you can use this fragment with sticky ends for ligation into your double-digested vector.

If you're also starting with a PCR amplification, it may be difficult to distinguish your product from the primers (I've also had trouble with cloning such small fragments). If you see anything on the gel, just take it and clone it into pJET first. If there is a restriction enzyme cutting in your product but not in your primers, that's great for checking what you got in pJET, else you will have to sequence.

Sound like your DNA insert coding for the shRNA behaves bitchy. The reason can be stable secondory structures like G-Quadruplexes in GC-rich DNA segments. Many other secondary structures are also possible that hinders efficient ligation. You can solve it by heating up the reaction batch WITHOUT ligation buffer and ligase to 60-70 °C to destroy the potential secondary structures and slowly cool down to RT. After that you can can add ligation buffer and ligase to your reaction batch. Then to make everything efficient as possbile you can let your ligation batch cycle between 12 °C and 22 °C overnight. The reason for this is that 12 °C is the optimal ligation temperature for hexacutting stick ends and 22 °C is the optimal working temperature for ligases; another reason is that by cycling the temperature you will induce convection flow in the reaction batch allowing continuous mixing :). Cycling the temperature is only a suitable method for sticky ends but not for blunt ends. If everything doesn't work then you can simply at you shRNA DNA coding segment in your plasmid by side-directed mutagenesis, containing 2 primer with sequence of your shRNA and of your corresponding plasmid region. The last method is 99% workinng according to my experience.

What I do in case of such small inserts: order the sense and antisense oligos with the appropriate overhangs, mix equal amounts, boil them in a water bath briefly, let the pot with water (and the oligos) on the bench to cool down slowly (this way they anneal anyway) and do the ligation (taking into account all other comments regarding ATP, competent cells etc.). This way it is normally a piece of cake.

Hi Atiqur,

I am curious about main purpose of your experiment. Do you want to clone this short fragment into expression vector to express it later on? Or your only need to amplify it to get high amount for other purpose? Why I am asking that because look at your fragment length, it is only 59 bp, it is not multiple of 3 bp. Maybe it is not the real fragment length, It could contain the extra cutting site,.. I assumed. So, whatever your purpose is, it is not good idea to insert directly very short fragment into such a big vector (~7 kb) like this. You should insert them directly into clone vector i.e pCR2.1 or pCR2.1 topo which is shorter (~ 2.6-3 kb) than your vector now. You can do it just after amplify by Taq-polymerase using the commercial kit, because you do not need to digest the fragment before inserting in those kind of plasmid (TA binding). Then you can do miniprep to obtain more plasmid contained your fragment. Once you get sufficient plasmid, you can digest with your desired RE, then insert those fragments into your desired vector now. By doing that, although you have to do one more cloning step, but at the end you are saving time with digesting, purifying, ligating very short fragment like that. And remember the ratio between fragment and vector in ligation reaction is 1:1 or 5:1 in molar (or in molecules more or less). In any case the fragment concentration should be higher than vector concentration.

Good luck.

the good thing is that, nowadays, Genscript can synthesize the fragment upto ~10 Kb. They also provide the services to clone your fragment into desired vector. So, I think best choice is ask them to synthesize for you. Few hundred (500-700 USD) for one construct.

Hi Atiqur,

I like these two ways of cloning an 59bp DNA into 6990bp vector. One way is as you described using the classic cloning method involving restriction digestion and ligation. In your description, it is not clear how you obtained the 59bp (assuming your are actually using DNA, not RNA) and whether it had ends matching those of the vector digested with HindIII and Xhol. If they have matching ends, there shouldn't be any problem using the ligation and transformation steps.

The second method is based on the gene assembly technology that neither need restriction nor ligation. NEB has a kit called Gibson Assembly Cloning Kit that will do the job. What you need to do is to synthesize a primer that have part of your 59bp DNA on each 5' end of the PCR primers that also have the 3' complementary priming sequence that matches the vector. There should be some overlap between the two primers. After PCR with hi-fidelity enzyme, you have the 59bp split at the end of PCR fragment. The Gibson Assembly kit will join the two ends together to a circular plasmid that can transform in competent cells. Hope you good luck!

You alter the temperature to 22o c or reduce the time from over night to 4 hrs

, or change vector insert ratio 1:1

good luck

Generate 2 complimentary oligos that include both sticky ends, and anneal.

Vector does not have to be dephosphorylated.

If background is a problem, postcut the ligation with a unique enzyme located between XhoI and HindIII only in the parental vector.

Alternatively you can order two complimentary oligos that have about 15 bp overlapping homology with vector on both sides, anneal, and do Gibson cloning:

https://www.neb.com/applications/cloning-and-synthetic-biology/gibson-assembly-cloning

The first thing you need is making cDNA out of your shRNA through reversed trancriptase PCR. Then doing regular PCR with appropriate primers (You can introduce your desired restriction sites to the cDNA fragment by adding their 6 nt sequence at the begining of your forward and reversed primers). Then later follow the cloning procedure through digestion, ligation and heat shock.

If you already have your primers without restriction sites, you can also use topo cloning. When you use taq polymerase for your PCR, this enzyme adds sticky"A" ends to the PCR fragments which can be cloned into a pJET plasmid containing various restiriction sites. You can look up the map online and check the possibility of findning your desired Restriction sites on the vector.

Thank you very much everyone for all of your good suggestions. I appreciate you all guys for putting your great efforts in solving my problem.

Dear Fernando Rodriguez Jahnke, thank you very much for your suggestion. You are right, I'm using corresponding DNA sequence. My insert has sticky ends which are compatible with the vector ends. Still the ligation is not working. Do you think I need to increase amount of vector and insert in ligation reaction as my vector size is 7 kb? Is there any possibility that my linear vector is twisted and has got secondary structure on it? If it is true, how I can solve it? Thanks.

Dear Thanh Nguyen, thank you very much for your suggestion. My main purpose of this experiment is to construct a clone containing my shRNA sequence. Then, after transformation and extraction of the clone vector, I'll transfect this clone vector into cell to knockdown a target protein expression by inhibiting the mRNA of target protein.

Dear Yinhua Zhang, thank you very much for your suggestion. I'm using corresponding DNA. I got the insert from a company and it has sticky ends which are compatible with the vector ends. For ligation, I'm using NEB T4 DNA Ligase. What else would be the problem for unsuccessful ligation?

Dear Koen Venken, thank you very much for your suggestion. I already generated 2 complimentary oligos that include both sticky ends and annealed. I'm using the annealed oligo which has 5' phosphate as well. Still not getting succesful ligation yet. What else would be the problem in unsuccessful ligation? Thanks.

Dear Mr. Rahman, I wrote you an answer how you may solve problems with secondary structures because this I think is your main problem. Click on "Show previous problems" to check my and other suggestions for your problem, I think you oversee them. Good luck!

Dear Dong-Jiunn Truong, Thank you very much for your suggestion. I appreciate you. How long do I need to heat up the reaction batch WITHOUT ligation buffer and ligase to 60-70 °C to destroy the potential secondary structures? Thanks.

5 minutes is more than sufficient, important thing is that you should slowly cool down (without using ice) and than add the buffer and ligase.

Please tell us if you succeed and what you exactly have done already.

This may be late but I had similar problems cloning my gene of interest. Your insert is quite small so it should ligate easier. I now ligate using 1:5 vector insert ratio in a total of 10 ul reaction containg 1-1.5 ul of ligase, keep at room temp. for 30 min then overnight at 4c. I have always got positive colonies since then. May be out of the protocol but works for me. Again you may try changing your competent cells. Good luck

By overlap extension PCR cloning. We are using it and it is very simple. You can find the protocol in the following papers:

-Overlap extension PCR cloning.

Bryksin A, Matsumura I.

Methods Mol Biol. 2013;1073:31-42. doi: 10.1007/978-1-62703-625-2_4.

PMID: 23996437

-Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids.

Bryksin AV, Matsumura I.

Biotechniques. 2010 Jun;48(6):463-5. doi: 10.2144/000113418.

PMID: 20569222

Good luck

Eladio

Hi Md Rahman: Sorry I did not see you question until today posted for me "Dear Yinhua Zhang, thank you very much for your suggestion. I'm using corresponding DNA. I got the insert from a company and it has sticky ends which are compatible with the vector ends. For ligation, I'm using NEB T4 DNA Ligase. What else would be the problem for unsuccessful ligation?"

Good question about an unsuccessful ligation: This is a most common question we got in our technique support, since the last enzyme used is T4 DNA ligase. However, a failure of ligation could be many factors. Since the reporter for a ligation result is often assayed by the number of colonies obtained by transformation, first thing is to make sure that you have high efficiency competent cells. 2nd, a control without insert is necessary for assessing ligation efficiency. 3rd, some impurities carried over from insert and vector is not uncommon and so don't use more DNA than suggested (in fact, too much DNA itself has some inhibition on ligation). For trouble shooting whether the T4 DNA ligase is at fault, a control ligation with known and repeatable efficiency is necessary. Good luck!