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Fugene 6 + serum free DMEM + problem


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#1 Iluvkittens

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Posted 22 March 2012 - 04:25 PM

I was transfecting HEK293T cells when I realized I put the tube with the wrong amount of DNA + Serum Free DMEM + Fugene 6 into the wells; there was not enough Fugene 6 when I double checked my calculations.

What if I had mixed the remaining necessary Fugene 6 (.5 ul/ well, I calculated) with Serum Free DMEM, let it sit for 5 minutes, and then dispensed it straight into the wells that already contain a Fugene 6 + DNA + SF DMEM? I'm guessing it wouldn't work, since it's not on any protocol, but what would it have done? Or would my results have been more messed up than they will be?

Does this question make sense?

#2 bob1

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Posted 25 March 2012 - 11:54 AM

Your results shouldn't have been any more messed up, but it probably wouldn't have worked - otherwise you could just add fugene + DNA to a well for a transfection. The reason why you set up the small volume of DNA/fugene is to allow the microsphere formation in a situation where there is lots of both the DNA and the fugene, thereby increasing the chances of the fugene microspheres encapsulating the DNA during formation.

#3 Iluvkittens

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Posted 31 March 2012 - 04:47 AM

Thank you for the response bob1! My mentor says I need to think more about how protocols work and "what if scenarios" since they can answer questions and you learn more about why some things are done the way they are.




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