Help: anyone who has co-transfected TWO different plasmids - - how much of each DNA to use? (Jul/20/2005 )
Hi
I used to think I will only need to transfect mammalian cells to express one protein, and have optimized (using GFP) how many ug of plasmid to use to maximise expression. But now it looks like I will need to co-transfect two plasmids to express two proteins (for co-IP experiment).
How would you go about deciding how many ug of each plasmid to use? Like I would use 3ug for one plasmid. So now do I use 3ug of each, or 1.5ug of each, or....? Do you all go through a complicated round of transfecting different ratios in 6 well plates, check expression with western blot, then scale up to dishes? Or is there some rules of thumb, some quick and dirty way to optimize, or some common ratio that works for you which I can use to start with? Will be grateful for any tips.
hi
i recently used the psi test and you need to cotransfect two plasmids. The ratio between the two ones varies regarding the exp you do. For my exp the first plasmid encodes an enzyme an the second one a siRNA. That's why it's recommended to use a &:5 ratio between these plasmids (a molar ratio!...)
And max weight is 3 to 5µg for a well from a 12well plate...
By the way, i would do a first exp in your case, regarding the best quantity for exp your protein in single transfection alone.
You'll get the amount of plasmid for both proteins. So try transfecting with these queantities and see... But you'll probably need to adjust the conditions before getting optimal results.
Hope that's a begining for you.
fred
Hi,
I always keep the total amount of DNA same. That means you should titrate the best ratio between the two plasmids. This will result in lower expression, but also in lower toxicity, so more cells survive.
Good luck:)
i recently used the psi test and you need to cotransfect two plasmids. The ratio between the two ones varies regarding the exp you do. For my exp the first plasmid encodes an enzyme an the second one a siRNA. That's why it's recommended to use a &:5 ratio between these plasmids (a molar ratio!...)
And max weight is 3 to 5µg for a well from a 12well plate...
By the way, i would do a first exp in your case, regarding the best quantity for exp your protein in single transfection alone.
You'll get the amount of plasmid for both proteins. So try transfecting with these queantities and see... But you'll probably need to adjust the conditions before getting optimal results.
Hope that's a begining for you.
fred
Hi Fred
So if I decide that 3ug is the optimal amount of plasmid to transfect one protein, what would be your suggestion to try for 2 plasmids- to transfect 1.5ug each? or like 1ug of one, 2ug of the other? (total still 3ug). Thanks
hi
if you decide to co transfect 3µg, i think you should calculate the mass of each plasmid to get a molar molar ratio.
That's said if you have 1plasmid 3kb and the other one 6kb, you should use 1µg of the "little" one and 2µg of the other one to get equal molar ratios.
Fred
Hello,
Is the difference of expression level really greatly affect the result of Co-IP? I am not sure should we use much much time to do the optimization for equal expression level of two proteins.
Should it be fine for Co-IP, if the WB showed that the two proteins, A and B, are really expressed in the cells?
I am young in this field, please give me some comment or corrections if you found. Thank you very much.
Patrick