Hi Harshraj Subhash Shinde,

the few times I remember checking  the ligation reaction by agarose gel didn't work. These are my recommendations:

1) Clean the insert before ligation

2) Cut the vector and the insert with two different RE to prevent self-ligation of the vector.

3) Make sure that the selection marker (antibiotic for instance) is fresh, at the right concentration and added after autoclaving the culture medium.

4) Use the primers of the insert and the vector primers flanking the insert in different colony PCR. By the way, colony PCR can be tricky sometimes.

Lastly, pick few colonies and do plasmid extraction then PCR to check that the colony PCR is working.

If it still doesn't work, try to dephosphorylate the linearized vector prior to ligation.

Hope it helps.

Hi,

You can check your ligation products by gel electrophoresis or PCR using plasmid primers across the insert but the number of ligation products and their low concentration makes analysis by agarose gel electrophoresis an impractical method. So, I think it’s not a good idea to confirm successful ligations prior to transformation. Your problem has two causes: incomplete digestion of the vector, and re-ligation of the cut vector with itself. At first, check that digestion of the vector goes to completion. I don’t know if you use directional cloning. Directional cloning result in non-compatible ends after digestion so vector re-ligation could be reduced (gel purification of vector after digestion is necessary) but it’s better to treat with phosphatase after digesting the vector (Remove the phosphatase with a spin-column. Gel purification of the vector is usually unnecessary, because phosphatase treatment will inactivate any extra DNA fragments.)

Following controls may help:

1. Transform the cut vector to determine the amount of background due to undigested plasmid.

2. Transform a vector only ligation reaction. The ends of the vector should not be able to re-ligate because either they are incompatible or the 5´ phosphate group has been removed in a dephosphorylation reaction. This control should yield the same number of colonies as befor.

I hope this help.

Dear Harshraj

Confirmation of ligation can be done using PCR. If you did cloning in a TA cloning based vector then use universal flanking primers (Vector based) to your insert. As a reaction you can take 1-2 ul of ligation product and can perform PCR. Do not forget to take a negative control that must be water and unligated vector both in separate reaction. Check the product on agarose gel. hope this will work definitely.

All the best

And another thing, Did you check the plasmid using restriction

Did you check the recombinant plasmid using restriction digestion instead of Colony PCR ? Because Colony PCR is not always authenticated procedure. You must do double digestion of your plasmid.

All the best 

What are the restriction you used for digestion? Your getting colonies but are not positive that inicates vector religation might be taking place. Try dephosphorylation of vector and then ligation. But while doing ligation follow the blunt end ligation protocol.to increase the efficiency of ligation. Before doing transformation you can confirm the ligation by checking it on gel provided that you must of linear insert , linear vector and circularize vector to confirm what's going on in your ligation reaction. However, alternatively you can check it ligation by pcr by using one primer from vector backbone and one from insert if it is directional cloning and not blunt end cloning.

Thank you all . It helps alot

@ Sachin I am cutting my gene using the Kpn and Spe I restriction enzyme. I am trying to clone my gene in pAUR123 vector. it is directional cloning. Thank you for answer. I will try your suggestions.

@Chet Ram. I was not getting positive colonies in colony PCR so i didnt proceed to plasmid isolation. Thank you for answer. I will try your suggestions.

Do SAP treatment, it will prevent religation of empty vector.

Thank you Debashis Dutta for answer.

Hi dear, with colony pcr this is the main problem that you can't confirm your recombinant bacteria, you do first extraction of the transformed vector that you have made recombinant and check on gel to confirm whether have you got the recombinant or not, 2nd dilute the pcr tubes because after cpcr you will get reptured cells, Yours gene will be so much encountered by the genomic DNA as well as plasmid itself, that yours primers will not be able to reach the target.

I read here about a strategy of cutting the ligated product with an enzyme that has been removed during the cloning. Since I ligate 10 ng DNA I would use 0.2ng enzyme.

I am doing that if my cloning is not successful (many colonies in the background and few colonies in the positive plate). I am curious to see if I have a ligated product.

My 2 restriction sites are very close to each other, perhaps sequential digestion is beneficial.

With Infusion cloning deP is not required, and the insert with Infusion Taq is suitable for cloning already without modifications, and the same is true for most synthesized oligos.

I do not routinely check a background plate, but I do when I fail a cloning. What is your background? In my case, the vector was looking linearized on gel, but probably there was a good amount digested only at a single site. This also after extending digestion time to 4 hours (sometime overnight is necessary for some enzymes).

You may want to check your cloning strategies. usually if I fail twice I change something (redesign primers, alternative approach etc).

I am able to look at someone's strategy and give inputs if desired.