# Ligation ratio problem - (Mar/12/2007 )

I am trying to ligate an insert with 1.6kb size into a vector with 8.7kb size. Obviously, the vector is much bigger than insert. Based on our lab protocol and some answers of this website or other websites, if the size of insert is less than the half of the vector, the mole or number ratio is between 5-10. I am confused how to calculate the gram and volume of insert or vector for the ligation mixture.

Someone said: (mass of vector/ lenght of vector)/ (mass of insert/lenght of insert) = ratio

I run the gel for 5ul of vector and 5ul of insert to compare the bands with marker size and find out the mass of insert and vector. the result was:

42 ng for vector band and 72ng for insert band( because the insert was 2times brighter than 1.5kb band of the marker):

then I did calculation based on above formula:

42/8.7/72/1.6= 1/10

but what is the volume of the insert or vector that I have to use for ligation?

Is this right to calculate them based on the total volume of the ligation mixture?

let's say 25ul total volume...then subtracting 0.5ul for T4 DNA ligase and 1ul for T4 DNA ligase buffer the remain would be23.5..now based on ratio 1/10 we have:

for vector: 23.5 (1/10)= 2.35

for insert 23.5(9/10)=21.15~21.2

are these caculations correct? please help me

Thanks alot!

Biomermaid

Someone said: (mass of vector/ lenght of vector)/ (mass of insert/lenght of insert) = ratio

I run the gel for 5ul of vector and 5ul of insert to compare the bands with marker size and find out the mass of insert and vector. the result was:

42 ng for vector band and 72ng for insert band( because the insert was 2times brighter than 1.5kb band of the marker):

then I did calculation based on above formula:

42/8.7/72/1.6= 1/10

but what is the volume of the insert or vector that I have to use for ligation?

Is this right to calculate them based on the total volume of the ligation mixture?

let's say 25ul total volume...then subtracting 0.5ul for T4 DNA ligase and 1ul for T4 DNA ligase buffer the remain would be23.5..now based on ratio 1/10 we have:

for vector: 23.5 (1/10)= 2.35

for insert 23.5(9/10)=21.15~21.2

are these caculations correct? please help me

Thanks alot!

Biomermaid

Hi.

The calculations for how much of what to add are correct, but I'm concerned about your calculation for how much vector and insert you have in the first place. Unless you have a densitometer, your calculations could be inaccurate. Why not just do a reading on a spectrophotometer at 260 nm? 1 OD is 50ug/ml. Do this and you'll know how much DNA you have to play with.

Then don't forget to try 5:1 as well as 10:1 ratios.

Thnaks, Ill try it and let you know.