Hi Reema,

so your vector is roundabout 10 kb, not too large, but not too smal...

Did you perform a positve control in the transfection experiment? Was it the same vector-size?

And you provided relatively few information: did you obtain any colonies in the transfection? or just a empty plate?

best,

Guido

Can you tell me the concentration of your backbone and insert which you are using for the ligation reaction and in which cells you are transforming the ligated product?

Hi Munich,

Thanks for your interest

My vector is 5.6kb (pGL4.17 vector, promega) and insert 5.2 kb, I performed positive control for transformation, also In my transformation I got few colonies but they were empty i.e not having insert.

Although not much clear in your query, but what I understand is that you are not able to get the proper restriction or expected restriction pattern after digesting the plasmid obtained from colonies picked up from the plate(after ligation). Following could be the problems:

1. Your vector might be not digested properly or when you cut your vector from agarose gel, you might be taking some uncut vector also. So after ligation you will get colonies of vector only.

Remedy: Cut your vector with enough of RE overnight. Next run it slowly and for long time in agarose gel. Then take only cut vector not uncut.

2.As evident you might be introducing your insert after cutting it from cloning vector(say PGEMT). here you need not worry about anything, just cut the insert out.

3. Check that your RE are fresh and everything is working in reaction. Also the same for ligation. Use fast ligase as it is more efficient than normal ligase.

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

Sorry for being lengthy in answer.

Good Luck

Hi Reema,

more or less i will agree with the rest of the suggestions. I have also found though that sometimes a 3 hour bench ligation may work better than one on ice. Also use always an excess of RE over your template to cut. Insufficient digestion may sometimes be an issue. Another tip is to try different analogies of the vector and insert. Try for example these ratios of insert:vector : 10:1, 5:1, 2:1, 1:1 and more surprisingly 1:3! You may just have to hit the correct ratio for the whole thing to work.

Also i would consider to subclone your insert into an easy cloning commercial vector so you don't have to repeat your PCR constantly. You just cut out a lot of DNA of interest from there and then you are fairly sure that you have a well digested insert.

Different people have different experiences so you may choose what suits you best.

Best wishes,

Nikos

Nikos and Tasaduq gave very good coments about the issue and with these tips you should manage it.

Best, Guido

perhaps you must review the oligonucleotide design. Do you add several nucleotide to de 5'end (3-5) for the enzyme support?

Another possibility other than merely cloning failure is that your insert could be toxic to the cloning host (I assume you are cloning into E. coli). If this is true, you are selecting for clones that have lost the plasmid, which is why you are only getting back empty vectors.

Since your vector is designed for high expression I would recommend trying a lower copy vector and perhaps recovering the bacteria at 30C instead of 37C and seeing how that works.

As a second to last resort you could try a strain designed for expression of toxic proteins, such as stbl1, 2 or 3 (https://www.lifetechnologies.com/order/catalog/product/11635018?ICID=search-product)

The last fallback position: just get it synthesized! Probably will be much cheaper in long run.

Good luck!

did you dephosphorylate your vector? If nothing works, blunt end cloning could solve some problems; ligation (at low temperatures for several hours, night, weekend; then add more ligase and incubate for up to an hour at higher temperatures...). Pick up to 100 clones, analyze in midi-preps... (sometimes only 3 of 100 are the right ones) --- it worked for me cloning very big K.O. constructs as 19 kb big plasmids of bluescript. Although I was the first to clone two CD44v K.O. constructs isogenic for the best ES cells (I became 6 months ahead in 2 years against 3 direct competitiors, two of them also postdocs - for sure, a questionable lab), I was not supported to make the partial CD44v K.O. mouse first.

Hello all.

Comments from Guido, Robert and Paul are quite right too. I guess if you try some of these methods, it will eventually work, don't get dissapointed easily, it's lab work after all and a lot of times it's not easy.

Good luck!

I have tried them different ratios of insert and vector, transformation controls with cut, uncut vectors too, performed blunt end cloning also. may be the toxicity issue is one thing which clicked my mind also.

well thanks to everyone

more suggestions are further welcomed

Hi gaurav,

For transformation I used 120 ng of vector backbone (5.6kb) and 350 ng of PCR product (5.2kb) in ligation mix. Actually i get colonies, they grow in fresh LB/Amp broth but they do not contain insert.

I remember, that it once took three postdocs in a lab for many months to just subclone one 3,5 kb, i.e. a normal-sized fragment of of the mouse CD44 gene, which I had cloned before and carefully mapped with dozens of restriction enzymes - all (.i.e. many!) other fragments worked at least after a few attempts - but this single fragment took so much time and effort. We never figured out the reason, but I shared my knowledge of mapping with my fourth colleague who did not have my map and lenght of introns, but she and the two questionable professors then published the genomic organisation of the CD44 gene (without me).

So, you should just try more, for example: digest several time your vector (add enzyme after a while), run a slow-running gel-electrophoresis and cut out the (cut) vector band from the gel, elute the DNA; you may also increase the insert ration, for example 10:1 (or reduce the vector). And then, you pick 100 or all colonies and check for inserts.

BTW, do all of your other clonings (if at all) work routineley fine? If yes, that could be a hint, that the sequence could be bad for the bacteria, but if you do not do much cloning, I think the reason is somewhere in the vector processing.

Try sure2 cells

Hello Reema,

My name is Robert Deyes and I work with Promega's Technical Support group. I second the point made earlier about toxic inserts. I have occasionally found inserts to be toxic to E.coli (ie bacteria just do not grow after transformation or they grow but the insert is excised out). One way around this is to do your overnight growth at a lower temperature (eg 30 degrees). You will get much smaller colonies that way (pin prick colonies that can only be seen by holding the plate up to the light). But they will be positive colonies nevertheless.

Another option might be to transform into a bacterial strain that is specifically designed for difficult-to-transform inserts/plasmids. As pointed out in an earlier response, Agilent Technologies sells SURE cells for this purpose (see http://www.genomics.agilent.com/en/Competent-Cells-Difficult-Cloning/Unstable-Clones/?cid=AG-PT-118&tabId=AG-PR-1218&Nty=1&Ntx=mode+matchall&Ntk=BasicSearch&N=4294967292+4294967234+4294967294+4294967244&type=baseSearch&No=0&Ntt=SURE).

Please let me know how your cloning goes. I would love to know how your experiments are going.

Thanks,

Robert

Robert Deyes

Technical Services Scientist

Promega Corporation

Tel 1 800 356 9526 Ext 1366 (USA)

Tel 1 608 274 4330 Ext 1366 (Long Distance, Toll Paying)

http://www.promega.com

Thanks Robert

I'll try the way you suggested, lets hope for the best.

Double check the quality of your insert/PCR product and use 20 ng of vector in the ligation mixture with inset:vector molar ratio of 1:3. If it doesn't work the first time assuming that all your reagents are OK, it would be good to look for another alternatives. Different ratios of insert:vector rarely help out. If you have a unique restriction in the insert, you can design a forward and reverse primers around that site and proceed with a two way sticky-blunt ends ligation. Make sure you add enough base pairs after the site for later restriction digestion. This approach helped me with ligation of large insert.

You could try to use E.coli cells for large plasmids, as STBL4, so the problem could be recombination or plasmid-loosing

Hi Belete and Fernando..

Thanks for your suggestions.

Recently, I tried cloning a 5kb amplicon in pJet1.2 vector and got few colonies, but as my insert is 5kb and vector is 3 kb so I shouls get linearsied band at 8 kb but I am getting bands at 6kb.

I have lineraised with RE in vector and also in Insert.

I also tried to digest with RE added in linker, but only one RE of linker is degesting and the other is giving uncut. ALso this RE is not present in either vector or insert.

I tried to get it sequenced, but three times sequencing failed...

I am getting hopeless now

Hi Reema, Maybe your vector is ligating to itself (hence the 6kb band after you linearized your clone). I would suggest using Antarctic phosphatase (https://www.neb.com/products/m0289-antarctic-phosphatase) on your vector after digestion to remove the 5' phosphate group. This prevents your vector from self ligating & will decrease the number of false positives.

best of luck! 

Valerie