Hi Wenxiu Ning ,

As you have not mentioned which particular vector you are using you may try changing the vector. Try increasing or decreasing DNA concentration. Is it not possible to digest both the vector and the DNA with same REs. You have performed this ligation reaction at 16 degree C or 4 degree C ?

Thank you very much!

This vector (8.2kb) is acturally pEGFP-C1 (4.7kb), which has already inserted one fragment I (3.5kb), What I do now is to insert another DNA fragment II (1kb) in front of the fragment inserted before.

There are only BspE1 and BglII sites can be used in the vector, but the fragment II(1kb) has one BglII site but not BamH I , that's why I choose these RE sites I mentioned before.

And I tried  different mole ratios of DNA:Vector from 3:1 to 10:1. Ligation reaction is performed at 16 degree c for at least 2 hours,

ALL failed.It is so hard...

I suggest to check whether your enzyme is cutting properly or not. Take any standard vector as a control to see if your enzymes are active. If single RE digestions are working on standard as well as your vector then ligation seems to be issue.

You should also check your eluted product on gel, since sometimes the nanodrop/vue gives wrong estimations.

How much nanograms of vector are you using? Since it is a big vector I suggest you take 50-100 ng of vector and 10 times insert. Try to do ligation in small volumes. I elute my vector and insert in water. Mix the required vector and insert volume and speed-vac-dry it, resuspend in 5ul of water and add 5 ul of 2X ligase. Ligation at 16 degree for 1 hr or 4-10 degree overnight should work. Plate the whole transformation mix on your selection plate with right antibiotic...:).

If still you don't see anything, may be your insert doesn't have the correct RE site, which you are trying, so check the sequence.

Wish you good luck...:)

Dear Wenxiu Ning, 

I would suggest to increase the incubation time from 2 h to overnight, if you want to perform your ligation at 16 degrees C.

Usually, the ligation is performed at lower temperatures (e.g. 16, 20 degrees) in order to reduce the molecular motion and to increase the contact time between compatible ends. However, at these temperatures the rate of the ligation is also reduced and thus requires prolonged incubation periods. 

If you can afford this, try your ligation overnight at 16 degrees C, if not - try for 1 or 2 h, but at 37 degrees C (or check the optimal temperature of the ligase you are using in the specification list). Good luck.

Thanks Harinder Singh and Stanimir Kambarev very very much. I will try 50-100 ng of vector and 10 times insert for at degrees C overnight. God help those who help me! Thank you  so so much! 

It may be a good idea to know the origin of your vector and insert. For example, if the insert is PCR amplified and purified from agarose gel that was run in TBE buffer. We had a ligation problem that was solved when we replaced borate containing buffer for TAE.

The above mentioned things should work. One additional thing is to order primers for your insert and design them in a way that they contain the restriction sites that are also present in the vector. Then you can cleave them with the corresponding restriction enzymes which makes the ligation potentially easier.

I would run a couple uL out on a 0.65% to check for a complete digestion. If it is all linear, then it could be that those enzymes add a 5' phosphate to prevent re-ligation to its self which would have to be removed by a phosphatase before it will ligate. I have always incubated my ligation at 4C overnight especially when it is finicky. If there is a lot of supercoiled product, then try increasing the amount of the enzyme and DTT. Hope you get it to work. It only takes one good colony in the end!

Thanks Nikolay Berezhnoy , Andreas Sturzenegger , Ginger Moore and all of you. I tried several times as you said these days, unluckly, failed. Maybe something wrong with te RE enzymes, but the sequence of the DNA-fragment-PRCblunt is right. It just did't work. I will try single RE disgestion  Thank you all so much. Good day~

Check the size of the insert, the orientations of the cut ends, incubation time, 3:1 vector:insert ratio, etc.,

Where actually do you find out that the procedure has failed? Is it that after bacteria transformation you find the plasmid without insert, or do you just not get any colonies?

If the Plasmid religates without insert, it might be also helpful to add a phosphatase after Plasmid Digestion, so it can only ligate together with your insert (which of course may not be dephosphorylated).

If you do not get any transformants, it might be helpful to check whether your plasmid-uptake competent bacteria are "sufficiently competent", so to either check a plasmid of similar size as a positive control, or if necessary use a strain which is optimized for uptake of large plasmids, since 9 kb are already quite a lot..

Hi Wenxiu,

1. Are there several extra bases outside of the RE sites (BspE1 and BamH I) of your insert (1 kb) band?

2. What kind of antibiotic are you using for resistance selection in the LB plates?

Attached is the map of plasmid pEGFP-C1, in case people are interested to know.

@Yuan-Yeu Yau, Christian Zerfass, I just got clones but no DNA fragment inserted. I will try to add phosphatase after Plasmid Digestion.Thanks a lot!

I am having the same problem. Were you able to solve it?

I have been struggling with the same proble.Have it worked?

Is your insert having stretches of redundant bases. If so the gene may form secondary structure and you may face difficulty in the ligation step. Please check the gene sequence. If this is the case then you may proceed with ligation independent cloning. I am saying this as I faced the same problem once.