Hello Mustafa,

I have ligated 12 Kb inserts in 4 kb vectors and then transformed using heat-shock in home-made competent cells with normal transformation efficiency like that of 10 to the power 6. It works, and with such crude methods also, it works.

Whatever I could gather from your query and replies, you have an 8 Kb digested insert, now a question, if these are staggered ends or blunt-ends, with one enzyme, meaning the insert can go in any orientation or is it directional cloning?

In any case, try the following:

1) phosphorylate the insert and dephosphorylate the vector,

2) Try to minimize the usage of UV, while cutting, eluting the bands. I have observed a strong effect of UV on the transformation efficiency, especially when the insert sizes are long. (It took me and my colleague 2-3 weeks and all sort of conviction form our boss that this can be a problem, before we actually listened to him!) This happens and I can tell you it is a major disaster, from getting no colony to getting a plate full of colonies, it was unbelievable, but true.

So, try to use an X-ray film and use preparatory wavelength and for as little time as possible, like not even a flash, but even less than that and not analytical one in the UV illuminator,

3) Try 1:1 vector to insert ratio and make sure that your insert and vector are properly and completely digested.

4) Go for prolong period for ligation, like overnight at 16 degrees.

Hope these suggestions help.

All the best.

Simran

maybe PCR amplify your insert with GW cloning extension and perform an LR reaction...if you do not need a specialize vector!

this way only the recombinant will growth..

Try to use Cold Fusion kit to clone your gene of interest into your vector. This kit efficiency is 99% and cloning procedure is very easy... It works on the principle of homologous recombination. SBI Biosciences sells this kit.

or...simply use blue light and a compatable DNA marker, and you will get it done. That way i managed to clone entire PV genomes (8Kb) with t4.

Hi, cloning efficiency is always improved when restristion sites are used in ligation procedure. If You can not follow the sticky ligation protocol try to make Your own T-vector stutaible for cloning the A-tailed PCR product. It should work better.

It's difficult due to the size. I recomend use at least 50 times of insertion. In addition you could use SAP for the vector (after digestion) to avoid religation. Anyway, maybe you should construct the clone in two steps.

Other option couold be recombination methods as Wanner.

How about a Gibson assembly?

Hi Andrei, doesn't work means I got colonies, I grew one in a 3 ml culture using the suitable antibiotic and purify DNA, cut the product with a known enzyme and ran a gel. the result was either no DNAat all or only the vector.

Thanks for your answer.

Did you try to check your ligation reaction by pcr before transformation? Is it possible that your problem is actually the transformation efficiency? Electroporation in my experience is more efficient for large plasmids.

Sorry, it was a typo. I meant antibiotic. My vector-only shows larger amount of colonies and it shows the vector (E. coli PBR322) on gel.

Thanks Francesco . I havn't checked my ligation reaction by PCR before transformation. Also, the transformation efficiency is just very good according to the positive control (the vector only).

Thank you.

What competent cells are you using? I used to clone large plasmids (20 kb) a long time a go and I know that the type of competent cell I used had a big effect on the transformation efficiency. Also, electroporation tends to work better for large plasmids than heat shock. NEB has a cell line (10-beta) that claims is better for large plasmids. Other than that, I agree with the above comments, using sticky ends and making sure your vector is dephosphorylated will help.

Hi Mustafa. Sometimes the problem is not the size of the insert and the vector. There are vectors which are specially difficult to clone due their structure. When I have that kind of problems, the first I do is optimize the reaction of the ligation by phosphorylating the insert and de-phosphorylating the vector even if I have cohesive extremes. The second, if I still can't cloning, is use a low melting agarose ligation. That method of ligation usually works well in difficult cloning. Good luck!

Thanks a lot Pablo García-Miranda. I'll try that.

Use clontech infusion

If you are working with two restriction enzymes, cut insert and vector with only one enzyme and ligate them together. Check on a gel if you get at least some product. Use that ligation product as PCR template for the second restriction site, digest the linear product with the second enzyme, ligate to make circular DNA, transform. Done.

If the first ligation doesn't work you can try to PCR insert and vector together instead of ligating them. If the second ligation doesn't work, transform the digested linear construct and see in E.coli did fix it correctly.

Hello Mustafa,

I have ligated 12 Kb inserts in 4 kb vectors and then transformed using heat-shock in home-made competent cells with normal transformation efficiency like that of 10 to the power 6. It works, and with such crude methods also, it works.

Whatever I could gather from your query and replies, you have an 8 Kb digested insert, now a question, if these are staggered ends or blunt-ends, with one enzyme, meaning the insert can go in any orientation or is it directional cloning?

In any case, try the following:

1) phosphorylate the insert and dephosphorylate the vector,

2) Try to minimize the usage of UV, while cutting, eluting the bands. I have observed a strong effect of UV on the transformation efficiency, especially when the insert sizes are long. (It took me and my colleague 2-3 weeks and all sort of conviction form our boss that this can be a problem, before we actually listened to him!) This happens and I can tell you it is a major disaster, from getting no colony to getting a plate full of colonies, it was unbelievable, but true.

So, try to use an X-ray film and use preparatory wavelength and for as little time as possible, like not even a flash, but even less than that and not analytical one in the UV illuminator,

3) Try 1:1 vector to insert ratio and make sure that your insert and vector are properly and completely digested.

4) Go for prolong period for ligation, like overnight at 16 degrees.

Hope these suggestions help.

All the best.

Simran

Thank you so much Simran.

Try Infusion cloning (Clontech) -I did 15 9-11kb fragments (they do need to be gel purified though) into a 4kb vector without any problems

Thanks Louise Elizabeth Bird