Hi Banismita,

When I get it right from your description you do a ligation of an insert in a vector and you are getting no colonies after transformation of the ligation mixture - is this correct?

It would be good to have some additional information.

1. what is vector do you use?

2. Which restriction enzyme/restriction sites do you use for cloning? Are these sticky site or do you blunt end cloning?

3. What insert do you have? Do you cut out the insert from another vector or do you perform PCR for your GOI?

When I am preparing the vector I do sequential digestion (if I use 2 enzymes I set up two reaction - 1 for each enzyme so if I use BamHI and EcoRI and I have i reaction which I digest with BamhI first and with EcoRI second and one reaction I digest with EcoRI first and with BamHI second. I do this to see if both enzymes are cutting properly - I control the efficiency on a gel between the first and the second digestion and after the second digestion)

After the digestion I dephosphorylize the vector with antartic phosphatase (but CIP should also work) but I do not clean up the reaction via gel but I usually precipitate with sodium acetate (3M) and 2,5 vol ice cold ethanol.

I do this to avoid UV radition during cutting out because this might damage your vector and in my hands this really helps to avoid this.

Due to the dephosphorylation the small linker sequence (I you have used 2 restriction enzymes) of your vector should be no problem as this is also dephosphorylized and should not lead to religation.

As you have no 5' phosphates at your vector (after dephosphorylation) you must have these at the sites of your insert. If you cut out your insert from another vector you should have these groups - however be fast when you cutting out your insert from the gel to avoid damage due to the UV light.

If you use a PCR product and you have added restriction sites to your primers you will have 5' prime phosphate groups after digestion, but if you are aiming for blunt end cloning you have to phosphorylate your PCR product.

I would avoid cutting out DNA fragments from a gel if possible (if you do this under UV light), however if I would have some more information I might can give you some more "targeted" advice - feel free to ask

In addition I would think that your vector concentration and your insert concentration are too high.

For a vector around the size of 5-6 kb 20 ng of good prepared vector should be enough.

Regarding the insert I have usually good results with a 2-3 molar ratio (this means if you have a 5 kb vector and 1 kb insert you would need 8 ng of insert for a 2 molar ratio if you use 20 ng of vector). To much DNA can really decrease your cloning and transformation efficiency.

Good luuck

absnece of clones can be du to

1: ineficient transformation (in your protocol you did not mention a heat shock.. )

2: ineficient ligation: a/Cip can be difficult to get rid off us antarctic shrimp phospatase instead of CIP (easier to inactivate)

b/ in my hands when insert and vector are both purified from gel, ligation can be inneficient could you have a strategy skipping the gel puification of at least one of the two molecules...

- have a look at using "Gibson assembly" instead of restriction and ligation (for example NEBuilder kit (but there is other brand)) it is much more efficient, works very well with unpurified DNA digestion (or PCR...)