I do a large amount of cloning and will encounter this problem from time to time. I have found that this almost always means that the vector is not cut to completion. I assume that you have cut your vector with two incompatible restriction enzymes. In this case, one enzyme can cut to completion while the other enzyme does not. The vector will then be a combination of single cut and double cut vectors. If you are not cutting a sizable fragment out of the vector then it will be impossible to determine if your vector is cut to completion with both enzymes by running on an agarose gel. When the vector is single cut it will easily self ligate in the vector only control tube ( this self ligation occurs much more efficiently than does the vector to insert ligation). In the vector + insert tube, the vector will still self ligate. However, a portion will also ligate to the insert on one side where the ends are compatible but will be unable to ligate on the other side where the ends are incompatible. This will essentially kill the efficient self ligation of the plasmid. Therefore, you will see more colonies for the vector only plate than you will see in the vector + insert plate. If there are some double cut vector in the tube then you may also get vector + insert recombinants and you may be able to find these if you test colonies on your vector + insert plate. The percentage will depend on the proportion of double cut vector that was in the tube and if one enzyme did not cut at all then you will have zero recombinant colonies.

If you are using two enzymes then I would suggest that you ensure that both enzymes are in fact cutting to completion by doing single digestions using the same reaction conditions that you are using in the double digestion. Phosphatase treatment will eliminate the self ligation but will not address the underlining problem of incomplete digestion. I hope that helps.

I've also experienced this (although rarely). In these cases addition of the insert seems somehow to inhibit ligation of vector alone or transformation efficiency (may be the insert preparation contains traces of compounds that have these effects or the insert by itself cause it). I suggest you anyway to go on isolating colonies and check your cloning. Indeed, when this happened to me the cloning was successfull!

Thank you very much. I will try to screen more colonies.

Has your linearized vector been dephosphorylated at ends?

My vector was not dephosphorylated.

Well, if your plasmid has got compatible ends (either sticky ends or blunt ends) that have not been dephosphorylated after linearisation, it will recircularize again efficiently without insert. (And most colonies in the "with insert" experiment may contain empty, recircularized, plasmid). Dephosphorylation is easy with any alkaline phosphatase (CIP or SAP). Just follow manufacturer protocols.

The vector is one side sticky and one side blunt. My problem is , almost equal number of colonies should be observed in test ligation also know ( vector+insert). I'm getting more colonies in control and less in test. Background is not that high ( around 200 colonies from the total transformation mix). I tried dephosphorylated strategy also. Result is similar in both the cases.

I´ve also experienced this as previously comment Cecilia Bucci. I´m agree with her that, in general, checking the colonies they are positives (at least 50%).

Hi. I would agree that there is something inhibitory about the insert. All other things being equal you would expect the same amount of background. 1) which enzymes are you digesting with? 2) are you heat inactivating the enzyme(s) after digestion? I highly recommend heat inactivation. Even after purification some enzyme can remain. 3) which ratios of vector/insert are you testing? In my experience, I find the sweet spot around 1:3-1:4. Higher insert ratios start to be inhibitory.

I have also experienced it before. Surprisingly, ~60% of experimental colonies had the insert. So it makes sense to check experimental colonies. Always good idea to use non –compatible restriction sites for cloning and plasmid which already had insert with flanking restriction sites of interest, so you know that your restriction enzymes worked. Sometimes you will not get double cut with restriction enzymes if they side by side on your MCS (though they will cut alone in controls). If it is the case, first cut with restriction enzyme which needs fewer nucleotides on the flanks of the restriction site.

Hello Jaya, when you use your control vector this is in its supercoiled conformation so cells can be easily transformed, in contrast your recombinant plasmid is linearised in ligation reaction so its transformation efficiency is lower. It could answer your question.

Additionally, here you have some tips:

* Use antarctic phosphatase to avoid plasmid recircularization after restriction reaction.

* Do not inactivate the ligation reaction, because inactivation decrease transformation efficiency.

Hope it helps

I am agree wiht Jacqueline. The main problem is your ligation's quality, Therefore, I think that the best resolution is inactive restriction reaction by alkaline phosphatase and heat.

Good luck.

Adding to the above suggestions, I suppose the problem could be of size... Insert + vector, the plasmid becomes 8.5 kb, which is not easily taken-up by the cell. So, the ligated products came in less number than the self ligated ones. The best way to deal with large plasmids is to go for electroporation. Sambrook's molecular cloning has protocols for making electro-competent E. coli.

The self-ligation problem (colonies in control plate) might be because one of your restriction enzymes did not work. Therefore, the cohesive ends from one RE digestion could ligate during ligation to give self ligated products. Another reason could be that the digested vector might not have been isolated from the junk, so the cohesive ends due to the fall-out would have recircularized your vector. To counter this, it is a good idea to test individual REs for their activity and after digestion, extract the digested vector from the gel.

For cloning larger inserts (from 3-5 kb) into backbone vectors ( ~ 5kb), it has been my experience that adding a "crowding agent" may help with the ligation. The vector:insert ratio is something that you can play around with, and set up multiple ratios at the same time. However, in the ligation reaction, certain protocols call for adding reagents such as PEG. The belief is that they serve to "sequester" the various components and keep them physically closer together, hopefully improving the ligation efficiency.

To minimize re-ligation of your digested backbone vector, as has been recommended above, treatment with a phosphatase (shrimp, antarctic, calf intestinal, etc) may also help. However, only treat your backbone vector. Do NOT treat the insert as the phosphate groups on either end are required for successful ligation. After restriction digestion, it's best to clean/purify (PCR cleanup kit/gel extract) your products before adding phosphatase.

Also, some people try to do ligation directly in the restriction digest tube, i.e. heat-inactivate the enzymes, then add ligase and other reagents. However, it's always better to work with clean products, so the quickest would be to use a PCR cleanup kit on your cut products first. These should definitely work for your two product sizes. Alternatively, you can gel extract the fragments, but this takes a bit longer. As ligation can be a very finicky process, I tend to always elute from the purification columns with water; that way, the only salts that are present in my ligation reaction are those that come from the ligase buffer components and enzyme. The insert and vector yields might be a bit lower, but they are cleaner.

Also, as has been mentioned, it's still worth screening potential clones on your recovery plates, even if the ratios seem off with respect to your negative control (i.e. vector alone). The most painless way is to do a patch plate of a large number of potential clones, then run a colony PCR using primers that should detect the presence of your insert.

I hope any of that helps. Just keep at it! Remember, all you need is ONE clone...

I think that what has been described is not dependent on vector supercoiling or usage of phosphatase. Indeed, the same vector (linearized with two different restriction enzymes) has been used in both reactions. Thus the amount of supercoiled vector (uncut) or of vector that it is able to ligate without the insert (vector cut with only one restriction enzyme) should be the same in both reaction. Also the fact that the insert is 3 Kb makes the cloning more difficult but does not justify why there are less colonies in the sample containing vector + insert compared to the one with the vector alone.

Decrease the insert amount....which restrcition sites have u been using....it might be possible that the ligase is busy ligating the insert -insert rather than insert vector...this depends on the restriction sites that one is using....(a single cut will lead to this proble.) the idea then is to use vector and insert in a ratio of 1:3 and could be varied....the last option cud be to make 2 fragments of insert and then clone serially...this is additional work however....larger the size of the insert to be cloned ..more difficult it is for ligase to ligate

Hi Jaya,

there has been a lot of good suggestions so far. We have seen this in our experiments and it is usually down to either the self inhibition of ligation by using too much insert or by a restriction enzyme not working efficiently and therefore you have single cut plasmid: both situations would reduce colonies on your ligation plate. We also run a vector only control (no ligase) to look for uncut plasmid. The situation is more complicated with a sticky/blunt combinations but from the stream it seems likely that you have too much insert present. A rough calculation of amounts: using 50ng of plasmid for ligation at a 3:1 ratio you would require ~ 80ng of insert.

Dephosphorylation of vector is a good idea and we have used PEG to increase molecular crowding (for blunt/blunt) but should not be necessary with this since the sticky end would create this situation anyway.

good luck

if you put 100 ng of vector put around 600 ng of insert (1: 3 molar ratio) Make sure restiction digestion is perfect and it has cut both plasmid and insert.

I do a large amount of cloning and will encounter this problem from time to time. I have found that this almost always means that the vector is not cut to completion. I assume that you have cut your vector with two incompatible restriction enzymes. In this case, one enzyme can cut to completion while the other enzyme does not. The vector will then be a combination of single cut and double cut vectors. If you are not cutting a sizable fragment out of the vector then it will be impossible to determine if your vector is cut to completion with both enzymes by running on an agarose gel. When the vector is single cut it will easily self ligate in the vector only control tube ( this self ligation occurs much more efficiently than does the vector to insert ligation). In the vector + insert tube, the vector will still self ligate. However, a portion will also ligate to the insert on one side where the ends are compatible but will be unable to ligate on the other side where the ends are incompatible. This will essentially kill the efficient self ligation of the plasmid. Therefore, you will see more colonies for the vector only plate than you will see in the vector + insert plate. If there are some double cut vector in the tube then you may also get vector + insert recombinants and you may be able to find these if you test colonies on your vector + insert plate. The percentage will depend on the proportion of double cut vector that was in the tube and if one enzyme did not cut at all then you will have zero recombinant colonies.

If you are using two enzymes then I would suggest that you ensure that both enzymes are in fact cutting to completion by doing single digestions using the same reaction conditions that you are using in the double digestion. Phosphatase treatment will eliminate the self ligation but will not address the underlining problem of incomplete digestion. I hope that helps.

I tried the same strategy with dephosphorylated vector. result is similar ( control more colonies and test more colonies). As suggested I will try to screen more colonies. Hope I will get positives. I always heat-inactivated ligase after ligation. As suggested, I will transform my ligation mix without heat inactivation. Thank you one and all for your valuable inputs.

I am agree with Pankaj Misra 1:3 ligation is best and it gives best results In Jaya's case I don't know what final volume she is taking. I will say that volume of the ligation reaction should increase and concentration of insert and vector should be decrese. Because, if insert and vector during ligation will not get free space for movement they will self ligate resulting you get self ligated colonies

I used 1:3 ratio. and also tried 1:5 and 1:10 also.

I think you should also check volume of ligation reaction, if you are taking 10uL reaction then you can try two other rections like 20uL, 25 uL with 1:5 ratio. During transformation you use only half of the ligation mixture. Good Luck!

Thank you Ashok. I will get back to you with the update results by Monday. I tried 20.0ul volume. 9 ul used for transformation and used 1ul is used for PCR to check the ligated product which didn't show any amplification.

Another problem with inhibition that I ran once into was too much glycerol in the reaction. Since enzyme preps are mostly glycerol to protect the enzymes, it my cause troubles. My rule of thumb was to not exceed 10% of the total reaction volume.

The other thing we usually did when we got only a few colonies was to test all of them anyway either by colony PCR or by prepping the plasmid not using a kit (the preps are quite raw then and contain a lot of RNA, but for checking on a gel, this works fine).

When dephosphorylating your vector, try using a fresh phosphatase and/or buffer.

For the ligation I use 2:6 and 2:0 for the control, in final volumes of 20ul.

I also had this problem and I agree with the explanation of Pankaj and Eric. Ratio 1:3 is the best.

Hi Jaya,

You've got some great suggestions here already. Phosphatase treatment will obviously help lower transformants from your control ligation. And yes, screening the experimental colonies will more often than not result in positive clones. The presence of insert does seem to change things a little bit and move the equilibrium away from self-ligation.

This does seem like a case of one or both of your enzymes not digesting your vector completely and therefore you get re-ligation on your control (+ligase) and inhibition of ligation when fragment is present. Another explanation could be that you are using too much insert in your ligation. I always start with a 3:1 ratio for sticky end ligations and if there are problems vary this.

We carry out single digests initially and take samples, then pool together and continue digestion with added RE's for a double digest (works if they work in compatible buffers). Analysis by gel electrophoresis is not always an indication of complete digestion as carryover can occur of undigested plasmid. Alk phos treatment is a good way of reducing this background as already suggested.

You could at each stage i.e for every single digest, check that it is complete by re-ligating ~50ng of plasmid (+/- ligase for 2hrs 37C) and then transforming (use 50ng of non digested plasmid as control). Do this at each stage to make sure your background is very low then proceed with actual ligation.

good luck

I agree with all excellent suggestion given above. Also try 1:3, 1:6 and 1:10 moloar ratios of V:I. I normally do not use more than 50ng vector. You can also try the following steps If you haven't done before

1. Transforming uncut vector in the competent cells checks cell viability and tests the antibiotic resistance of the plasmid. Plate on media and media plus antibiotic.

2. Linearized vector tests for background due to uncleaved plasmid. Should be

Thank you very much for all your suggestions.

I could trace out the issue associated with my question. In this cloning, I was trying to isolate the insert( 2.9 Kb) which was cloned in the vector of 2.7 Kb in size. The insert is contaminated with few vector molecules, which is finally giving more number of colonies in test. As my control ligation is not having the insert , I'm getting less colonies in control ligation. Prepared insert into two fragments to avoid the parent vector contamination and did ligation. This worked fine and the clone was confirmed.

Hello Jaya,

I'm facing the same problem these days ... could u plz explain more how u solved the problem? I also have insert (1100bp inside vector 2500 bp) and i need to take it and insert into another vector but every time digestion and ligation not successful and i get empty vector after transformation.