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I come up short

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#1 Iluvkittens

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Posted 23 February 2012 - 03:06 PM

I have 2 six well plates.  The first row has (in triplicate) 550ng PGL3-Basic.  In the second, I have 100 ng of (in triplicate) PGL3-Control and PCDNA3 (450 ng).  In the third row I have PGL3-Control again.  In the last, I have ASC (400ng), PGL3-Control (100ng) and Caspace-1 (50ng).

I have to place 300ul of Fugene and media mix into each aliquot of DNA.  For the PGL3-Control, I need 300ul x3, and the PCDNA3 I need 300 x 2.

But I can't seem to figure out how much Fugene I need to add to the media, and I always come up short.  Help!!!

#2 bob1

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Posted 23 February 2012 - 04:09 PM

Fugene usually works at a ratio of 3:1 based on microlitres of fugene: ug of DNA, so for your first row you would use 550 ng =0.55 ug x 3 (wells) = 1.65 ug DNA x 3 (from the ratio) = 4.95 ul fugene.

#3 Iluvkittens

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Posted 23 February 2012 - 05:46 PM

Thank you so much.  I was trying to measure all my DNA and fugene needs at once.  Of course I could break it down by row!  Thanks so much!!

#4 Iluvkittens

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Posted 24 February 2012 - 02:55 PM

Okay, I must be stupid or something... same scenario as first post.  But if I have to add PGL3-control plus Caspase-1 + ASC, should I try to find out how much Fugene to use per DNA sample (The PGL3-control Fugene amount, and then the Caspase-1 amount, and then the ASC?  Or is it okay to just add the total ng of DNA and then find out how much Fugene to use?  This also has to be in triplicate.

#5 Iluvkittens

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Posted 25 February 2012 - 03:44 AM

#6 bob1

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Posted 26 February 2012 - 04:09 PM

Fugene is being used to transfect all the different DNAs at the same time, therefore, add them all together, multiply by the number of replicates (triplicate =3), then multiply by the ratio...

#7 Iluvkittens

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Posted 22 March 2012 - 06:19 PM

I'm not sure how I missed this reply... thank you so much Bob1!