The answer depends entirely on your method of cloning. Unless you are using a blunt end cloning strategy or digestion with a single restriction enzyme, self-ligations should not be a problem. There are also excellent ligation-independent, recombination-based cloning strategies (Gibson cloning, Gateway cloning) that eliminate the problems of recovering empty vector.

Can you please post more information about your cloning strategy?

Use a phospatase to dephosphorilate the plasmid (preferentialy SAP, you can also use CIP) and USE for example T4 PNK for phosphorilation of the insert, prior to the ligation. That will reduce self ligation to almost 0.

The answer depends entirely on your method of cloning. Unless you are using a blunt end cloning strategy or digestion with a single restriction enzyme, self-ligations should not be a problem. There are also excellent ligation-independent, recombination-based cloning strategies (Gibson cloning, Gateway cloning) that eliminate the problems of recovering empty vector.

Can you please post more information about your cloning strategy?

For blunt end cloning we use a cut-ligation protocol that works really well. You add the insert + uncut vector backbone together with restriction enzyme, ligase and ligase buffer and cycle the reaction for 20-30 times at 37°C (the temp of your enzyme) for 2 min / 25°C for 5 min, then heat inactivate. Afterwards add fresh enzyme + phosphatase and incubate for another 30-60 minutes. You should get a high number of correct colonies after that.

Only thing you need to make sure is that the insert does not contain the enyzme you use in the cut-ligation.

Choose the one based on your expt.

1. Use phosphatase treatment if its involving Coheshive/ Sticky ends.

Note: Some phosphatase are difficult to Heat inactivate!

2. Using a gradient of Vector : Insert ratio, may yield less self ligation. There is no fixed rule but only guidelines!

May be you should mention more details - Conc. of Plasmid, Restricion enzyme used...

Use insert DNA concentration as 10 times more than plasmid

first calculate that how many times the vector is larger than insert. based on this set 1:6 vector to inser ratio. For eg. if plasmid is 5000bp and insert is 300bp than it shows that your plasmid is 16.6 times larger than insert. if you are using 50ng of digested plasmid than the conc. of insert for 1:1 would be (50/16.6) 3.01ng and for 1:6 it would be 18ng. I have personally experienced that 1:6 is the best ratio for ligation.

I agree with most of the guys here, you should start by fixing a molar ratio (1:3 or 1:6 are a good starting point). In my experience dephosphorilation of the vector with CIP works great!

SAP is always better than CIP as it can be heat inactivated.

Double digestions with sticky ends also can give religation of the plasmid when one of the enzimes works slower than the other, or specially when the restriction site is close as far as some enzymes do not like cutting in the edge.

Thank you very much for all the suggestions. We are performing all these approaches. It is a blunt end cloning, the plasmid was digested and dephosphorilated before the ligation. We used the proportion 1:3 and we didn't obtained sucess, but the next step will be use 1:6 and we will see. Thank you, all the suggestion were helpful.

If you are working with very large inserts (2kb or more), lower molar ratios (1:1) might be preferable to higher ratios (avoid multiple copies of inserts). Make sure you avoid excessively high total DNA concentrations, too, as this may promote vector-to-vector non-productive ligations.

My insert has 300 bp, now we are using 1:5. I hope we will have good results. Thank you!

In that case, if you switch to SAP, and use 5x excess of insert, you should be successful.

If you are using a single enzyme digestion, phosphatase treatment could solve the problem. otherwise, you could reduce the vector molar ration with respect to the insert

Hi

Self ligation is often observed problem and it happens due to sticky ends of the vector. The better way to prevent self ligation, as said already by many members, is to go for phosphatase treatment. The very process makes the ends blunt and prevents self-ligation. But in addition to difficult deactivation of the phosphatase latter, less concentration of the dephosphorylated vector is also a problem. However, it works fine.

From your question it is clear that you are getting vector colonies only. You need to be sure that your vector is completely cut, especially if single RE is used. Some RE are slow cutters(e.g., SpeI). Provide the restriction reaction with excess of RE and do it overnight. Later run the products of digestion on agarose very slowly and for possibly long time. Excise only the restricted part from gel carefully.

Before setting up ligation be sure to know about the concentration of the vector and insert and set up ligation with proper controls as:

V-I-L (vector minus insert minus ligase)= No expected colonies

V-I+L= expected colonies from vector only

V+I+L= expected colonies from vector+insert only

Cheers

https://www.researchgate.net/post/Problem_in_getting_positive_clones?ev=home_person_add_comment_question_target

Hi,

I generally do ligations with 50ng of vector. After digesting the vector, I run it on a gel, cut it out of the gel and purify it. Then I measure the concentration and I only SAP treat the amount I need (or somewhat more).

So if I do 2 ligations (the actual ligation and the negative control), I SAP treat ~110ng vector and then heat inactivate the SAP. I have tried adding SAP to the restriction digest, but that wasn't as succesful.

The mimimal volume for my ligations is 10ul, otherwise your DNA concentration in your ligation mix may be (too) high.

Good luck!

The vector to insert ratio mentioned by others is surely an important factor. However, so is the total DNA concentration. The whole process can be viewed as competing chemical reactions. Basically, we want combination of DNA ends from other molecules to successfully compete with self-combination. At very low total DNA concentrations the chances for two molecules to "meet" in the solution are very low, regardless of the insert to vector ratio. However the ends that could self-ligate are kept close together nevertheless.

There used to be a formula in the Maniatis cloning manual many years ago which also took into consideration total DNA concentration. It was based on mass action kinetics of chemical reactions and disappeared from later editions. Of course, most protocols call for the correct concentration anyway and there is no need to explicitly calculate this.

@Andreas Binder

I am interested in your 'cut-ligation' protocol for blunt end ligation you mentioned above. But I don't understand the step "cycle the reaction for 20-30 times".

could you please break down further what you mean?

@Yudistira Wahyu Kurnia

The cycle here means PCR cycle... we can do incubations in the PCR thermocycler also. Say here you can create a program with two steps first 37 degrees for 2 mins and second 25 degrees for 5 mins. The cycles will be set to 20-30.

@Jaswinder Kaur Saini

In this procedure(cut-ligation) after the Restriction and digestion do v need to add ligase and phosphatase with the respective buffer for 30-60min incubation ?

After this process shall we directly go for transformation or v need to do any further purification before transformation?

@vijaya Gowri Esvaran

In reference to the cut-ligation procedure suggested by Andreas Binde, let me expand it a little.

Step1

For blunt end cloning, insert, uncut vector backbone, restriction enzyme, ligase and ligase buffer all were added in a single reaction and incubated.

Result = vector with insert ligated in it, self ligated vector and some uncut vector

Step2

Now to remove self ligated vector and uncut vector, the above used restriction enzyme (not ligase) was added again along with phosphatase in the same reaction and the reaction was further incubated for 30-60 mins. As the self ligated vector and uncut vector have intact restriction sites, they will get linearized and phosphatase treatment will prevent self ligation. In this case both the enzymes can be put in the same reaction. Alkaline phosphatase usually works fine in ligation buffer or any other similar buffer.

But compatibility of an enzyme with the reagents should always be checked otherwise. You can follow the charts available online which list the performance of enzymes in different buffers.

After this the reaction can be directly used for transformation. Purification before transformation is optional.

@ Andreas Binder

Can you please explain your experience a little more? The cycles and the temperature all this is not clear to me. i also facing the same problem.

Treat vector with phosphatase and /or increase insert to vector ratio (6:1)