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Dilution of 18s. How much?


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#1 Podge

Podge

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Posted 10 November 2011 - 08:35 AM

Hey everybody,

establishing RT and qPCR in my new lab.

Today I had my first go:
I used 1ug of RNA for my cDNA synthesis (in 20ul reaction volume) and diluted this 1:10.
I used 2.5ul per 25ul qPCR reaction using Primers for bActin and got Cq values around 29, the variance is very high though and thus I want to try 18s tomorrow.
I'm just not sure how much I should diute my cDNA to have nice Cq values (15-25). Has anybody got an idea what a general dilution for 18s is?

Furthermore I am thinking of diluting my cDNA only 1:5 for my other genes of interest to get levels more in the range of 20. Does this make sense?

I appreciate your help!

Best regards.

Podge

#2 Podge

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Posted 11 November 2011 - 08:10 AM

Had a testrun today and tried 1:10, 100 and 1000. 1in1000 gave me Cqs of ~20, so I'll stick with that then.

Podge




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