# 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?

-pBS-

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

-Bomber-

QUOTE (Bomber @ Jul 5 2005, 02:58 AM)
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.

hello, bomber

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

-pBS-

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

-Bomber-

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

-donisaid-