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Transfection with lipofectamine

transfections lipofectamine

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#1 KhaleesiDany

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Posted 23 October 2015 - 01:38 AM

greetings

 

I am about to do a transfection with lipofectamine on the protocol it says: For each well in a 24 well dish: a.)In one microfuge tube add I ug DNA and 50 uL OptiMEM (if none is available, you can use DMEM or RPMI without FBS or antibiotics)  b.) In a second microfuge tube add I uL Lipofectamine® 2000 and 50 µL OptiMEM. 

 

From this i thought that i just need to spec my dna samples and use the appropriate amount to make 1ug dna for example for my total dna to be 1 ug in concentration i add 7.90 ul of dna. so dont i from there just directly add 50 ul optimem as in step a of the protocol, and then do step b and mix and eventually add to wells?

 

i was looking at my supervisors protocol and she adds water to the dna mix to make it to a volume of 12.3ul, then she adds 37.69 ul of the optimem to dna  and then 50 ul of the lipofectamine:optimem solution. My confusion is at the water step why first make it to 12.3ul? 



#2 bob1

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Posted 23 October 2015 - 11:50 PM

Your supervisor's protocol sounds like a classic example of a received protocol... i.e. one that has been set up (by someone else) and is followed blindly. I suspect the 12.3 ul thing is that they were initially using 2 or more plasmids, one of which required 12.3 ul to make 1 ug of total DNA, so all others were also diluted to that volume for consistency. There is no need to do the 12.3 thing, just use the DNA volume you calculated.







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