The procedure looks alright, so it is a bit difficult to guess where the problem is. That is what I do when I have problems with clonings.

Digest smaller amounts of DNA overnight; like 3-5ug of vector and 1-2ug of PCR product. This way you are sure to fully digest.

Deactivate the vector digestion at 65C for 30min and dephosphorilate the vector, THEN gel purify. This reduces the number of purification steps and the loss of DNA. Follow your kit's recommendations to enhance elution efficiency.

I would avoid gel purification for the PCR digestion, the fragments are so small you can eliminate them with a normal spin-column purification. Also, check that the extension you added to the PCR product to introduce restriction sites are long enough to allow efficient digestion (some enzymes need up to 6 bases from the end on the molecule to cut efficiently).

Sometimes I had better results eluting in Tris buffer (without EDTA) instead of water.

Quantify the DNA concentration.

Ligation ratios should be molar rather than by volume. I usually follow this general rule of thumb: 25fmol of vector + 3x25fmol (1:3) or 5x25fmol (1:5) of insert . I don't think you need to go as high as 1:10.

I don't know why you add ATP to the ligation mix, but just follow the enzyme recommendations for that. You can try a longer of overnight incubation to increase the efficiency.

Finally, ligations are usually transformed into 50uL of competent cells, 400 sounds a bit too much. You can try 2-5uL of ligation per 50uL of cells. If you have access to other cell types, you can try TOP10 or something else in addition to DH5a.

I hope that helps, good luck!

The procedure looks alright, so it is a bit difficult to guess where the problem is. That is what I do when I have problems with clonings.

Digest smaller amounts of DNA overnight; like 3-5ug of vector and 1-2ug of PCR product. This way you are sure to fully digest.

Deactivate the vector digestion at 65C for 30min and dephosphorilate the vector, THEN gel purify. This reduces the number of purification steps and the loss of DNA. Follow your kit's recommendations to enhance elution efficiency.

I would avoid gel purification for the PCR digestion, the fragments are so small you can eliminate them with a normal spin-column purification. Also, check that the extension you added to the PCR product to introduce restriction sites are long enough to allow efficient digestion (some enzymes need up to 6 bases from the end on the molecule to cut efficiently).

Sometimes I had better results eluting in Tris buffer (without EDTA) instead of water.

Quantify the DNA concentration.

Ligation ratios should be molar rather than by volume. I usually follow this general rule of thumb: 25fmol of vector + 3x25fmol (1:3) or 5x25fmol (1:5) of insert . I don't think you need to go as high as 1:10.

I don't know why you add ATP to the ligation mix, but just follow the enzyme recommendations for that. You can try a longer of overnight incubation to increase the efficiency.

Finally, ligations are usually transformed into 50uL of competent cells, 400 sounds a bit too much. You can try 2-5uL of ligation per 50uL of cells. If you have access to other cell types, you can try TOP10 or something else in addition to DH5a.

I hope that helps, good luck!

Thank you so so so much ~

Thanks for telling me a lot of your experiences.

Some things you said that I have never thought or noticed, then I consider the major problem is the "Base pairs from terminus of RE cutting site" , which caused the failed ligation.

I'll do the ligation cloning again. If the result is expected, I will response to you.

Thank you so much, sincerely !

Two things to think about:

First, try using less DNA, as you may be overloading the reaction. Your vector and insert need to anneal and then ligase has to find it to close the gap. If you have too much DNA floating around, the limiting reagent becomes ligase binding to your DNA. You don't need that much for successful ligations.

Second, you did not say how long you ran the ligation reaction for, I second the suggestion by Andrea and lengthen the ligation reaction. I too have found the best results to be overnight at 4 C.

Also, as a side note, CIP is not necessary with directional cloning when you are cutting with two different enzymes, and sometimes that may reduce efficiency, although that's not the problem here since you have 0 efficiency!

Some restriction enzymes require a minimum amount of extra bp to its left or right side to be able to cut a PCR fragment. I had the problem once where I did not add enough extra bp at the PCR ends, the enzymes would not cut and had to clone the PCR product in a cloning vector first and then cut it out to ligate it to the intentionally vector. Make sure you have around 5-10 extra bp to the left/right of the recognition sequences. Also some fragments might be toxic to the cells when expressed, you might want to try a different cell strain. In order to check the ligation you can set up a large amount of ligation and run part of it on an agarose gel after the ligation incubation, you can try various time points of the ligation. At t=0 you should see just vector and fragment bands, then over time that will convert into a smear or distinct bands of ligation products of the size of vector+fragment.

Dear all,

I have constructed my interested clone 3 months ago.

Thank for your(s) kind recommendations!!!

Sincerely,

Mason

Where was the problem?? What then helped you for successful ligation & transformation?