quanitifying single stranded DNA using a ds standard - (Nov/05/2006 )
I'm using qPCR to quantitate the titer of my virus, which is a single stranded DNA virus (AAV). I think I have made my standards correctly, but they are coming out differently from the standards made in our lab several months ago (and which I cannot find the calculations, as they were made by a postdoc who has since left).
So here is the main question, since the standards will be double stranded plasmid DNA and my viral genome is single stranded DNA, do I have to multiply the sample by two in order to normalize?
I think the calculations control for the double stranded nature of the DNA but I am not sure. Here are my calculations:
260: 0.574 and 280: 0.475 260/280= 1.21. My plasmid is 5892 bases.
5892 bp x 1.096x10^-21 g/bp = 6.46e-18 g/plasmid copy
1e10 copies x 6.46e-18 g/copy = 6.46e-8 g
6.46e-8 g/ 5 ul = 1.292e-8g/ul
V1= [(0.029e-6 g/ul)(200ul)]/1.292e-8 g/ul
V1= 448.9 ul
so I added 200 ul of my stock to 248.9 ul of TE. I then diluted 1:10 for 1e9 copies in 5 ul.
It seems that the g/bp conversion should account for the double stranded nature of the standard. So I don't really see the value in multiplying my samples by two, although this is what is typically done in the lab.
Here is a simpler way of doing..... just modifiy a bit of your PCR profile.
additional step : 70oC 5 min
Ta 30 30s
72oC 5 min
follow byu your actual PCR profile.
the 70oC incubation is to open up secondaray structual on you ssDNA.
Ta is annealing temperature for your primer to anneal on the target gene.
anf 72oC is primer exstantion..... therefore will convert your ssDNA into dsDNA.
As you can see, there is no denaturing step in the additional step, thus your standard which is in plasmid form, would not be open up yet. Got it
Remember, use slidely more dNTP in the reaction as the additional step will use up some dNTP.