I have two plasmids: plasmid A (8434bp)and plasmid B (12,082bp). I want to do co-transfection of these plasmids in different ratios of molarity by keeping the total DNA amount same (1.5ug). The desired molarity ratios between A: B are 5:1, 1:1 and 1:3. The concentration of plasmid A and B is 580 ng/ul and 1265 ng/ul, respectively. Can anyone tell me the formula to achieve required ratios within 1.5ug of total amount of DNA?
For most DNAs (those with near average G-C composition)m wt is just under 660 per bp. If your two plasmids have similar proportions of each base then the ratio of molecular weights will be 1: 1.433 (A:B) so for a one to one molar mix in 1.5ug you need to add 1.433/2.433 x 1.5ug = 0.883ug of B and 1/2.433 x 1.5ug = 0.617ug of A. I am sure you can move from these amounts to calculate the volumes. Similarly you can move from 1:1 to 5:1 if you add 5 x as much A or 1:3 with 3 x as much be. The easy way to get the correct proportion is to ask what volume you would use if A was 5 x as dilute or B three times as dilute. Calculate the volumes to be mixed on this basis and then work out the total volume and concentration and for each mixture and therefore how much contains 1.5ug.
Hi Nasir
I do not have a direct formulae to help you but I can an suggest an easy way to calculate.
First I would say that one need 0.69 times less weight (8434/12082) of A than B to get equal number of moles.
Now suppose the amount of B needed is X ng; so to get 1:1 molar ratio yielding total amount of 1500 ng, the equation would be
0.69X + 1X= 1500
X= 1500/1.69 ng
X= 887 ng or approx 118 fmol of B
Now amount of A needed is= 1500-887= 613 ng or approx 118 fmol
Similarly for 5: 1 ratio the equation would be
5 x 0.69X + 1X = 1500
4.45X = 1500
X= 337 ng or approx 45 fmol of B
Now amount of A needed is= 1500-337= 1163 ng or approx 223 fmol
I hope it will solve the issue.
Thanks
Cheers
Prafull
Hi,
I would use NEB bio calculator. Or any other online tool, such as one provided by IDT.
Good luck
hi Nasir
Did your transfection worked??
i am facing similar problem , when i transfect small plasmid having GFP signal , efficiency is 60% in HELA cells. However, once i cotransfect with another plasmid which is 8.5 kb length , efficiency drops to 10% when i see GFP positive cells. How should i improve my transfections. Do i need to add more transfection reagent for larger plasmids???? i am using viafect from promega
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