Working out PEI:DNA ratio for transfection - (Oct/26/2008 )
I am an undergraduate doing transfection for the first time but I am really confused on how I calculate a range of PEI:DNA ratios for optimising transfection of BKH 21 cells.
How do I know how much PEI to add and how much DNA? I think it is 2ug of DNA but all I have is DNA from a miniprep so I don't know how much is in it?
I am completely lost and any papers I find are not helping. Thanks in advance.
Dont worry, I will take you through step-by-step.
First, try to run a gel with a series of DNA with known concentration (like 0.25, 0.5, 0.75 and 1.0 ug), then spot your sample to the next well. You run the gel, stain it with EtBr, take a picture of it. This will help you to get an estimate of what DNA concentration is in the mini-prep by comparing the band intensity to the known samples. If your miniprep has DNA and RNA bands, you need to get real concentration of nucleic acid in your sample, because RNA carry negative charges as well. (read Molecular Cloning to determine which bands are which) Next, you read OD260 of your sample that has been diluted in TE. Then use the formular 1 OD260= 50 ug/ml DNA to calculate the amount of nucleic acid in your sample. Its best to use pure DNA, but for some quick experiment, dirty DNAs may also be used, but you need to use the total nucleic acid conc in this case.
Second, you need to know what size of the culture plates are you going to use to determine the amount of DNA each well to use. 2-4 ug DNA seems to be the amount for one well in a 6 well plate. Go to internet and find a table of surface area for each multwell plates and calculate how much DNA should you use each well, if you are not use 6-well plates. Manufacturers typically have these data on their website.
Third, use a ratio of 1 nmole DNA phosphate to 10 nmole PEI nitrogen to prepare the complex.
Good luck to your first transfection experiment and I hope you find fun doing science!
Thank you so so much!