Hi Mohammad,

sometimes, there can be problems, if the two restriction sites are located very close to each other. Some enzymes require a restriction site to be located a certain distance from the end of the fragment (general rule 6bp), otherwise they cut with a low efficiency. you might try to read something about troubleshooting:

Recognition site may be too close to the end of the DNA fragment (for example this: https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments).

From my experience once i had a similar problem, when EcoRI did not cut after digestion with another enzyme. You could try to cut your vector first with EcoRI and then with XhoI.

Hope it'll help, good luck.

Andrea

Hi Mohammad,

now i found another pdf:

http://www.staff.ncl.ac.uk/p.dean/Biosuite/Cleavage_close_to_end_of_dna__NEB_.pdf

and it seems, that EcoRI should not have problems, but instead XhoI can give you some difficulties to digest. so maybe try to first cut with XhoI and then with EcoRI

HI Andera

thank U for your Answer

but I said in my question that I digest my vector firstly with XhoI and after that with EcoRI, even I do an Ethanol precipitation after each digest but the results was a lot of self ligated again.

Hi Mohammad,,

i had works about double digested from amplified product and vector before, but, I used pBI121 as a vector, EcoRI and BamHI as a enzimes.

my experience, amplified product and vector was digested with EcoRI and BamHI all at once and incubation on 37 C just 3 hour. and then must purified from gel, you must make sure that estimate of digested product size was it's true,,especially PCR product,,

and for ligation, all of componen,(vector, DNA insertion, T4 DNA ligase, 2X ligation buffer, H2O) was incubate on 4 C ON (over night).

For transformation, you use electrophoration or heat shock?, and transformation into E.coli or Agrobacterium or the other ?

i hope it's can help you,,

Self ligate colonies usually come from incomplete digestion of plasmid. I suggest you use fastdigest enzyme from Fermentas, not conventional restriction enzymes. Since XhoI and EcoRI of fastdigest could work in the same buffer, fastdigest buffer. Though fermentas boast that 5 min is enough for cutting plasmid. For me, I usually digest for 3 hours.

Do you digest your DNA in large enough volume? sometimes that helps. Another thing you could try, is to dephosphorylate your plasmid after digestion, like this you should avoid self-ligation.

Hi friends

thank U

@Dilla

Dila I think my ligate reaction and transformation process has not serious problem because I use of a cutted control plasmid for my expriment that show the ligate and transformation are OK. however I've used of DH5 and heat shock for transformation and I've put my ligate reaction in 4'c over night.

@ Andrea

I think you are right about that one of my enzyme isn't working properly and seem it is xhoI, so I want to use of R buffer for firstly xhoI digest and after Ethanol precipitation use of EcoRI buffer for second digest. also my digest volume is 30 ul. if this did not work I'll be using dephosphorylation in next step.

Hi,

I remember a similar issue with the XhoI enzyme in our lab and as far as I remember XhoI from differerent manufacturers can give different results. So if can get hold of Xho from NEB, for example, run a similar restriction digest in parallel with your previous mix. Definitely digest sequentially and don't forget to dephosphorylate your vector for at least an hour.

Good luck!

Jenny

Hi guys,

I found that using Gibson cloning is almost always much easier and robust than ligation. For instance you won't have self-ligation. My lab is using this method for more than 2 years with excellent results. You can read more here: http://en.wikipedia.org/wiki/Gibson_assembly or in the original paper by Gibson et. al. (http://dx.doi.org/10.1038%2Fnmeth.1318).

Hi,

finally I cloned 300 nucleotide fragment in both orientation in pHannibal vector,

I think my problem was restriction enzyme buffer,I've realized that when you are working with XhoI , you should use Buffer R for double digest reaction because xhoI does not work in tungo 2x buffer, This is in contrast with the recommendations Fermentas site.

http://www.thermoscientificbio.com/restriction-enzymes/xhoi/

thank you all

I am not sure what other people have suggested above, but you could try amplifying your PCR product by cloning it into pGEMT or something similar prior to digestion. This will give you a much greater amount of purified cut 300kB fragment and may improve your efficiency. Plus if you digest it out of pGEMT and cut the small fragment you will also confirm that your fragment has been correctly digested.

Hi Mohammad, I was having similar problems with pKANNIBAL so, I cloned my PCR product in pGEM and I sent to be sequenced, in the results of sequensation I found that my PCR product realy haven't the sequence for the restriction enzimes (despite it is in my primers sequence), now I'm still trying to solve this problem.

I too am having trouble with self ligating plasmid, while using the EcoRI and the XhoI restriction sites, like you have had. I have checked and ruled out my plasmid purification and ligation techniques, and whether the restriction enzymes have activity, with appropriate controls. To date I have tried normal and fast digest fermentas and NEB. I have looked at the pHannibal plasmid map

http://www.huayueyang.com.cn/product/276835493

and like the plasmid I am using, pChlamy_4, it has the EcoRI and the XhoI restriction sites int the MCS, next to each other and only seperated by 1 base pair.

I believe this is the issue and that the plasmids are not being cut properly by the XhoI as this has the problem of needing several base pairs up and down stream to cut properly. To date I have only tried simultaneous double digests but will now try sequential digests with separate samples in which either EcoRI or XhoI cuts first then the other. I will let you know which works.

These are the websites which detail the activity of the enzymes in relation to base pairs around the restriction enzyme recognition sites.

https://www.neb.com/-/media/nebus/files/chart-image/cleavage_olignucleotides_old.pdf?la=en

https://www.neb.com/-/media/nebus/files/chart-image/cleavage_linearized_vector_old.pdf?la=en

I will also use the shrimp alkaline phosphatase to prevent plasmid self ligation