I would try a longer digestion time for your vector, I normally do 3 hours to overnight. That way there is less carry over the the uncut vector. What kind/brand of T4 ligase are you using? You could try a longer ligation time (depending on the kit instructions). Also, the ng of vector seems a little low. A general guideline is to use 200 ng of vector per every 3 kb of vector size. For the insert, use a 3 molar ratio of the insert:vector. For example, if your vector is 3 kb and your insert is 1 kb, use 200 ng of vector and 200 ng of insert. That way you have three times as many insert molecules as you do vector molecules. I usually set up a 20 ul ligation reaction and then use 5-10 ul for the transformation.

Thanks t4 ligase also thermo scientific.

I calculated the amount of insert I added with an online tool.

But thanks I will try that out!

It seems that you are putting too much insert. Also what do you mean by "denatured proteins" - is that heat inactivation , if yes then 75oC for 20 min will be ok. I would use 1/10 of the ligation mix for transformation (20s @42oC) if your cells are competent you will have plenty of colonies. I would also recover for longer time (30 min or more) and be gentle on the Kan concentration (it is a low copy vector). Hope that helps

I meant i heat inactivate them under exact same conditions you mentioned. I will try less insert than.

Dear Felix,

can I just ask if you have some extra bases between the restriction sites in your PCR primers and the end of the primers? For example NdeI needs extra >3 bases to cut effectively, see https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

I also think your insert amount is not right. Using a 1:3 vector:insert ratio for sticky ends it should be 20.5 ng insert with 50 ng vector or 40 ng insert with 100 ng vector.

You could try a slightly lower incubation temperature (22C) and a longer incubation time (1 hour) for your ligation.

Kemas Aurino

Hi, I will be cloning into a NdeI/NcoI digested vector. What do you mean by lower volume of ligation mix (1ul?). Do u mean enyzme+buffer?

Or does this mean final volume used for transformation?

Felix Scharge So you get 4 colonies in your negative control. That means that one of the enzymes did not work properly.

Try this:

Incubate your vector with one of the restriction enzymes

1 µg vector DNA

1 µL restriction enzyme Nr. 1

5 µL buffer

4 µL rSAP (NEB)

up to 50 µl ddH2O for 1 h at 37°C with shaking in the same reaction tube.

Then heat-deactivate and gel-purify the vector.

Set up a second reaction using the second restriction enzyme and repeat reaction together with phosphatase rSAP as above. Purify using column kit (no gel purification).

Then ligate as usual.

The negative control should not have any colonies and only plasmids containing the insert should survive on the plates.

Good luck

Himanshi Bhatia Hi, Did you solve the problem ? I am doing NdeI and NcoI digested vector for ligation. I did 3 times with different parameters with no luck.

Nothing on the plates and nothing no the control plate with no insertion. Any protocol can help? thank you!

Rather than digesting your PCR products directly, you could try sub-cloning them (pTOPO or similar) then digesting (this cloning relies on topoisomerase, not cut and paste so it's a quick way to capture a gene or gene fragment). It's an extra step but it let's you verify that your enzymes are cutting your insert. Another thing you could try is a different ligase. NEB sells a Quick Ligase that works in 5 minutes at room temperature.