From my experience there might be something wrong with the digestion or you are not using enough insert. Run a control digest with a plasmid that will release a fragment when digested with the same combination. 

Also, is the insert coming from PCR or also from a digest? If it's from a PCR then maybe you should scale up, if it's from a digest then I would re-evaluate your approach and look  for errors in the protocol, "cut and paste" cloning is pretty straight forward, you may be overlooking a flaw in one of your steps. 

Send a detailed protocol of what you are doing and I'll look at it.

corey

https://www.thermofisher.com/iq/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/cloning/cloning-troubleshooting-guide.html

Dear Aditya

2 main questions?

1) The vector control plate was prepared using the same vector concentration than the ligases or yuo just trasformed the empy vector with out dilute it with a water volume corresponding to the insert?

2) Which kind of vector? Pet? and in this case JM109 or JM109 DE3? Because some times your problem happen if you try to clone a toxic gene in a strain able to express the protein. In this case if the promoter is not well repressed, the production of the protien can prevent the colony growth also if the ligases happen, and therefore you have a false negative result.

In this case you can try to:

change E.coli strain.

change the plate medium  (e.g with addition of glucose for t7 promoter plasmids) to bettere repress the expression.

good luck

Manuele

Hi there,

What do you call vector control plate? Is it digested vector alone without ligase or digested vector alone with ligase? If the former, I agree that you should at least get the same result with the insert... If the latter then it means the clones you see come from self ligation and that the presence of insert prevents this self ligation but doesn't lead to recircularization of the plasmid.

Thanks everyone for the suggestions. 

Vector control means the same amount of digested vector along with ligase with NO INSERT. Self-ligation is not possible here because both ends have different restriction sites (BamHI and XhoI), so the colonies I am getting on Control plates might be due to undigested vector.

Vector backbone is 326 which is a plant expression vector system having 35S promotor. It can not express in E.coli as per my colleagues suggested.

I have cloned other genes in the same vector but never faced such problem.

Hoping for the best, please suggest more

Thanks again everyone

When you do a double digest for your vector, can you see a released fragment? If not, are you sure both enzymes are functional? If you have another plasmid with an insert already in it that you could cut out then use as a vector, you would at least answer the problem of self ligation.

After digestion, separate digested plasmid on agarose and excise digested plasmid from gel( Undigested plasmid will migrate at different position). Even for double digestion, You should use CIP to prevent recircularisation of single digested plasmid. 

you cannot distinguish double digested and signgle digested plasmid on gel because both REs are in MCS( very close).   Most of the cases, main problem of cloning is the in efficient digestion of plasmid.  

Hi again,

your control plate doesn't discriminate between undigested vector and self ligation event. To get sure about the event responsible for the clones you observe you should systematically run 2 controls: plasmid alone (clones will result from undigested plasmid) and plasmid alone with ligase (clones will result from either undigested plasmid or self ligation event).

Your restrictions sites may not be compatible with JM109. Try DH5 alpha comp cells.