I would suggest a 1:3 vector to insert ratio.And you can try setting up ligation reaction overnight on ice. Or follow the usual protocol of 22 degree for 1 hour, followed by 18 degree for 12 hours.

Hello Sandeep,

1. The concentration of vector after gel purification is too low, have you checked the purity (260/280 and 260/230 ratios).

2. Have you done a sequential RE digestion/Double RE digestion. I would suggest to use at least 2-3 micrograms of vector for RE digestion.

3. Use 1:3 vector to insert ratio for ligation, keep vector amount 50 nanograms/ligation. You can calculate the amount of insert by using online calculator at http://www.insilico.uni-duesseldorf.de/Lig_Input.html

I would suggest a 1:3 vector to insert ratio.And you can try setting up ligation reaction overnight on ice. Or follow the usual protocol of 22 degree for 1 hour, followed by 18 degree for 12 hours.

You gave us information about concentrations, but not on the final amount. How many ng of vector and insert did you finally use? As you surely know the number of free ends is the most important parameters for setting up a successful ligation reaction and you have to calculate the mass of insert required at several molar insert:vector ratios in the range needed for typical ligation reactions.

@ Dheeraj Chaudhary , the 260/280 ratio for vector DNA is very poor, 47, but for insert is 1.76. @ Gianluca Occhi, we are using 10 ng of vector and 30 ng of insert.

never worked with less than 40 ng of vector, but I'm not sure DNA amount is the problem. I would suggest to start with a novel plasmid digestion with higher quantity of plasmid DNA. With Gel DNA Recovery Kit obtaining larger quantity of DNA is not that difficult.

On the other hands are you sure that your bacterial cells worked properly? Positive controls?

Thanks to all for valuable suggestions, I have started plasmid purification and will double digest again for higher concentration.

Vector:insert ratio of 1:3 has always worked for me.

Generally I use this formula for ligation purpose i.e. Concentration of vector (ng)x Insert size (kb)x3 /Concentration of Insert (ng).

Very nice suggestions by all. To add up, I have couple of points

1. The V:I ratio, 1:3 has been recommended in many literatures considering the bigger vector and smaller inserts for example 0.5-1kb insert and 3-4kb vector as recommended by pGEM-T easy system, and it works. However, as the insert size increases, the ratios has to be changed in other way round by reducing the concentration of insert. i.e. the ratio of 1:1 works well when the insert and vectors are almost of the same size.

2. The total concentration of final ligation mix per reaction of 10 or 20 ul are to be decided based on the efficiency of the ligase enzyme. Excess DNA per reaction will hinder the reaction. Also, enzyme to buffer ratio must also be as per recommendation.

3. Use recommended amount (units) of ligase per reaction. Add ATP and PEG if doing blunt end ligation.

4. Go for CIAP treatment, if going for ligation with same restriction sites at both the ends to avoid self ligation of insert or vector.

5. One can pool 2-3 ligation reaction mixtures of 10 or 20 ul each to enhance the probability of successful ligations. But do not go for transformation with more than 5ul ligation mix/100ul comp cells. Because, the higher amount of ligase have inhibitory effect on transformation. Instead, go for multiple replicates using the same ligation mix.

Good luck

BASAVARAJ

Thanks to all for valuable suggestions but right now the real problem I'm facing is how to increase vector and insert DNA concentration after double digestion. It is not going beyond 6 ng/ul even after digesting 40 ug/ul of vector DNA.I think it is the problem of gel extraction kit I'm using, can I try P:C:I extraction after double digestion or suggest me some other cheap method which can work well. Basavaraj Y.B. Sangeeta Rathore Sebastian Schmitt

Sandeep Kumar

Your concentration is too low.Try to purify in more amount.This will ensure your ligation will surely work.

Generally 1:1,1:3,1:5 work depending on your Insert and vector size.

Gud luck

Dear, there are lots of factors that could help.

-Always start digestion with about 3microgram so you have enough to work with.

-Ensure the two enzymes are working by running single digests too

-Ensure the distance between the two sites are reasonable so any cut can easily be noticed.

Try different ligation ratios

Cloning could be annoying sometimes, best wishes

digested vector concentration today was = 26 ng/ul

my digested PCR product concentration today was = 13 ng/ul

so for a 1:1 ratio according to pmol end, and for a total volume of 20 ul I used

10 ul (260ng) vector

7.5 ul (98ng) PCR product

2 ul Buffer

1 ul T4

0.5 ul water

Dear Shahjajhan,

It´s good that you have now more vector and insert, but in you reaction you used more vector than insert.

I always worked with a 1:3 or even 1:4 vector:insert ratio, but never used ng for this, I prefere to calculate pmol, because what is important is the number of molecules, and making the calculation of pmol you are considering the size of the plasmid and insert.

To do this calculation I use the formula: pmol= (ug DNA/ul) (1000000) / (# pb DNA) (660).

I also suggest to include a restriction control, that is that you make a ligation reaction without insert, to be completly sure that your vector is properly restricted. And I also prefer to do a 16hr ligation reaction.

Hello Sandeep Kumar

I would like give you my experience too

I think would be good to start double digestion around 1ug to 3ug even 5ug since after gel purification it will be in the order of ng, this point is critical.

You can use the ratio V: I, 1: 1, 1: 2, 1: 3 as mentioned before, although most of the time I use 1: 1. I always work with the final mixture of ligature by reaction 10 ul. 1 ul of 10x T4 buffer, 1 ul of T4 enzyme and 1 ul of NFW as a premix, the rest 7 ul will depend on the concentration of V or I and the proportion used.

Another important thing as Chiara Gamberi mentioned is about your competent cells.

Gud luck

Go for electroelution, bit time taking procedure but you will recover most of the double digested vector/insert. The yield from electroelution is way more than any of the kits available in the market.

How much DNA should be taken for ligation depends on the competence of your cells as well. You can vary it in the range of 50-150 ng for vector.

Use ligation calculator for the amounts to be taken for vector and insert.

Usually 1:3 works well. DO check the vector and insert on the gel before ligation, (sometimes just going by nanodrop makes it complicated) whether it reflects the same what is being estimated.

all the best!

Hi Sandeep,

Also look for your transformation protocol because it happens usually that we do not get colonies due to some errors in transformation protocol.

If you like to ask anything about transformation protocol then you are welcome.

in my experience success was always depending on DNA quality and almost never on DNA quantity.

Thus: be sure that both enzymes have cut the vector. make sure that both enzymes have cut the PCR product e.g. by first cloning it into a vector and release it from there. if

if both insert and vector have perfect ends, you can throw them together in almost any ratio and the ligation will work

1:3 DNA: Vector is optimum . Its depends also the Size to insert and vector. If you ate using any Kit follow the protocol.

It may also depent on type of your expression vector or expression construct for ligation of DNA. It is recommended 100 ng of your totla DNA and expression vector, to ligate your inset into, about 1:3 ratio. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.