# concept of the molar ratio - concept of the molar ratio (Jul/05/2005 )

hello to everyone,

i am going to ligate the insert of 1kb into vector of 3kb. and i want to take the molar ratio 1:3, is there any one who can guide me in calculating the molar ratio. and i am using the plasmid bluescript, i use the .2-.3M pot acetate and double vol of abs alcohol and overnight incubation for precipitation. i want to know whether i am correct or not.

and what other salt can we use for precipitation and their molarity?

thanx in advance

Hi,

to calculate the molar ratio you can use the following consideration:

A base pair(!) has a molecular weight of 660 g/mol; so if your vector is 3 kb long, that means 3000 bp x 660 g/mol = 1980000 g/mol. If you use for example 100 ng vector for your ligation this would be 5,05 x 10 exp -14 mol vector.

(100 ng divided through 1980000 g/mol gives you mol).

If you want to do a ligation in the ratio of 1:3 (vector : insert) that means you have to use 3 x 5,05 x 10 exp -14 mol insert. The only thing you have to do now is to calculate how many ng vector are 3x 5,05 x 10 exp-14 mol with the same consideration as above : one base pair = 660 g/mol.

This calculation is not 100% correct but it general you can use it.

If you don´t want to calculate that much just try some different volumes betwenn insert and vector. Usually this works too.

Your precipitation sound ok to me.

Good luck,

Cheers

to calculate the molar ratio you can use the following consideration:

A base pair(!) has a molecular weight of 660 g/mol; so if your vector is 3 kb long, that means 3000 bp x 660 g/mol = 1980000 g/mol. If you use for example 100 ng vector for your ligation this would be 5,05 x 10 exp -14 mol vector.

(100 ng divided through 1980000 g/mol gives you mol).

If you want to do a ligation in the ratio of 1:3 (vector : insert) that means you have to use 3 x 5,05 x 10 exp -14 mol insert. The only thing you have to do now is to calculate how many ng vector are 3x 5,05 x 10 exp-14 mol with the same consideration as above : one base pair = 660 g/mol.

This calculation is not 100% correct but it general you can use it.

If you don´t want to calculate that much just try some different volumes betwenn insert and vector. Usually this works too.

Your precipitation sound ok to me.

hello, bomber

thanx for your response,

i understood the way, how we calculate the molarity. but the line u have written is not clear to me.5,05 *10 exp -14 mol

could you please explain this to me.

and i want to the plasmid 2 microgram. then how much insert should i t take.

thanx......

Good luck,

Cheers

Hi,

5,05 x 10 exp -14 means 0,0000000000000505 mol.

Two micrograms sounds very much to me. I usually take something around 50- 100 ng vector, but anyway.

2 microgram (µg) are ~ 1,01 picomol (pmol) for 3000 bp vector.

That means for 1:3 (vector:insert) 3,03 pmol insert.

3,03 mol insert are (if insert is 1000 bp) ~ 2 µg.

Good luck

Cheers

You can use a direct relation, i mean:

Insert + Vector = plasmid

1kb + 3kb = 4kb

? + ? = 200 ng

200*1/4=50ng

200*3/4=150ng

in this way you get a molar ration of 1:1