Hi Parimala, You can give a try on the followings-

1. After digesting your vector, run on a gel side by side with the uncut vector to see if your vector is really cut with the restriction enzyme.

2. Try to tritrate, 3:1, 6:1, 9:1 (vector: insert ratio) which gives a positive result.

4. Which ligation kit you are using? If T4 DNA ligase, then overnight incubation of 'ligation reaction' at 4 degree C sometimes increases colony number.

If still your problem persists, you may use In-fusion cloning kit from CloneTech company which always gives brilliant result and very easy to use.

Best wishes for you :) 

Q: "Once i had got colonies through regular ligation method but almost after incubation for 18-20 hours. How reliable are these colonies?"

Just take a few colonies and do miniprep for PCR or 'colony PCR' to check whether your PCR product has successfully ligated to the back bone (vector). When you do PCR, you can design a primer from the backbone and one primer from the insert (your PCR product).

Dear Yuan- Yeu Yeu,

It is possible to do 'colony PCR' to check if the colonies are really having the insert. Here, it is easy to design the primer from the insert how can we be sure the primer from the vector?

Thank you

Hagos

Hi Hagos Mohammedseid Juhar,

1. Some vectors have a complete backbone sequence available. If so, you can design a primer from the backbone sequence outside the insert region.

2. If a vector's whole sequence is not available, there are some existing popular genes (ex. selectable gene--kanamycin-resistant gene, hygromycin-resistant gene, bar gene,....), selectable marker gene (GUS, GFP,......), promoters (35S or others),.......on the backbone. You can design the 'backbone' primer from those genes because most of these gene sequences are well known.

Thank you every one for your advices.

@Sharmin- I have tried doing 1:3 (Vector: Insert) ligation. The digested product on gel does show a clear band of desired size. I perform gel extraction after 2nd digestion and use it further for ligation. I use NEB T4 DNA Ligase and incubate my mixture at 25degrees. I have done it for 15mins, 3hours, 6hours and overnight (12hours). I have almost done 13 times and i'm still not able to figure out my mistake (if any). I also tried doing a detour by using TA cloning. the positive clones i excised the insert using same enzymes and then i tried putting into my vector, but still it doesn't ligate. all the colonies i have screened until now show up to be false positives. What should I do now.

@Yuan- I didn't do colony PCR but I did screen those colonies using Restriction digestion. They didn't have the insert.