While cloning I am only getting self ligated vector not any clone. I had digested Genomic DNA of my strain with Eco R1 as well as vector (PGEX). After ligation at 4C, i transformed the ligated product (with coontrols )but i am only able to got self ligated vector not any clone.
Hi Versha,
when you open a vector with just one enzyme i recommend you to dephosphorylate the vectoe ends with alkaline phosphatase (CIP). This should eleminate self-ligation of the vector.
The next thing you have to consider when ligating a fragment is the molar ratio of these in the reaction (usually you will find some hints in the ligase manual).
best,
GAD
You could try changing the ratio of vector to DNA. Maybe the amount of DNA was too low.
Hi varsha,
Here I want to suggest you something:
1. You have told that you have digested genomic DNA. I want to be clarified whether you want tell about whole genomic DNA or the PCR product amplified from the genomic DNA. Look there should be lots of EcoRI sites within a whole genomic DNA which need not to be within your desired gene i.e. amplified PCR product. So, you can easily understand that its very difficult to get your desired clone if you are following the first one.
2. Its always better to use two different restriction enzymes to get clones because it will give you more specific clones and the number of self-ligated vectors also be very less due to double digestion.
3. Last, if you are restricted to use single enzyme then follow the procedure mentioned by Guido.
Best of Luck !!
As Guido said, it is really difficult to clone without dephosphorylation step when using a single Restriction Enzyme.
Calf Intestinal Alkaline Phosphatase (CIAP) is really good, but it is difficult to remove or inactivate. For me, it works using CIAP for 30 min 37ºC followed by sequential phenol and chloroform:isoamyl alcohol (24:1) extraction and ethanol precipitation to completely remove it.
Other option is using Shrim Alkaline Phosphatase, that becomes inactivated after 5 min at 4ºC, but I prefer the first option.
Good luck.
As mentioned, phosphatase of the digested vector and removal of the phosphatase prior to the ligation is extremely important. And when you perform ligation, you should always include a parallel control reaction to which you add water instead of insert DNA--i.e. with only vector. This will tell you what the background of simple plasmid vector reclosure is. If you compare the number of colonies you get with this control and with the +insert reaction colonies, you will have an estimate of the proportion of "empty vectors" among the +insert colony set.
Also why are you doing ligation at 4C? At that temperature, the ligase will be hardly active and you may be recovering only vector plasmid that was uncut to begin with. 12 or 16 degrees overnight or just room temp for a shorter time are more typical reaction conditions for ligation.
As others have mentioned, the most likely reason you aren't getting any inserts is due to not removing the phosphate groups on the ends of your digested vector. Additionally, as already mentioned, whenever possible, you should use different enzymes to clone with (ideally 2 enzymes that are active in the same buffer so you can perform a double digest).
Question though, are you trying to make a genomic library or are you trying to clone a PCR product? There is a substantial difference and a little more information from you would help to resolve your problem further. It sounds like you are simply digesting genomic DNA with RI, and then shotgun cloning those fragments into a vector. Successful genomic library construction can be tricky sometimes to get adequate representation of the genome, and you also have to factor in things such as what host strain you are transforming, etc.
Try also to use higher concentrations of DNA. Digest lots of Vector, run on gel then extract it. If you are using 2 enzymes, test first if all of them are working properly
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