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I am interested in transfecting HEK293 cells with a BAC. One paper suggests using 24 ug of DNA to transfect a 55-cm^2/10 ml culture plate. I am using an assay optimimzed for a 96 well plate so I am interested in scaling down the amount of DNA.

If I consider by volume: I will be going from 10 mL to 100 uL, scaling down by a 1/100, suggesting 0.24 ug of DNA.

If I consider by cell number: I will be going from 10^7 cells to 10^4 cells, scaling down by 1/1000, suggesting 0.024 ug of DNA.

If I consider by cell concentration: I will be going from 10^6 cells/mL to 10^5 cells/mL, scaling down by 1/10, suggesting 2.4 ug of DNA.

What factor should I scale down by? I want to say that it's volume but I don't really know why.

Thank you for any help or suggestions!

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    $\begingroup$ Are you using lipofectamine? They provide tables to sort of help streamline the process: 3000 reagent, 2000 reagent. $\endgroup$ – CKM Jul 13 '16 at 16:59
  • $\begingroup$ Hi - No, I'm not using Lipofectamine but I did see the table online. I'm wondering why they decided to use volume instead of cell # or conc which seems much more intuitive to me. $\endgroup$ – molecular.me Jul 13 '16 at 18:32
  • $\begingroup$ Well if you think about a plate-based transfection, only a certain number of cells will viably fit into a well, and you want them in a replicative state. So you try to get them 90% confluent, which is easier visualized in a 6-24 well format, but for 96-well other groups have worked out the best cell/well in their protocols already. Consensus is at around 50,000 cells per well. So the cells numbers are actually standardized for the plate format. The only question that remains is how much volume of reagents will work with said cell counts! $\endgroup$ – CKM Jul 13 '16 at 19:17
  • $\begingroup$ Wait, I don't think that answers the question. If the cell # is standardized, then amt of DNA transfected should be scaled down according to cell # not volume $\endgroup$ – molecular.me Jul 13 '16 at 20:33
  • $\begingroup$ If you know how many cells will give you confluence in a 96-well format, you just scale down on the size of the well. So 1:100 or 0.2 ug DNA. $\endgroup$ – CKM Jul 13 '16 at 21:01

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