Measuring the amount of PCR product - (Mar/14/2006 )
HI!
Is there any technique for a fast measurement of my DNA concentration in my PCR-product.
Poblem: In my DNA extracts are:
1) different concentartions of polymerase inhibiting substances
2) presumably much DNA that is not amplyfied (also in different concentration)
so think it does not make sense to measure the DNA concentration of my extracts.
I heard there is a dye (name?) staining only larger pieces of double stranded DNA, so that i don't measure my dNTP's. Are there additional methods?
thanks for your advice
hi,
one solution:
-PCR your samples
-electrophoresis and ethidium bromide
-use one of the new MW rulers knowing the exact mass for each fragment in a given loaded volume --> standard curve
-scan or CCD
-count intensity for each band of interest (ImageJ freeware) and relate to the standar curve
Seb
another solution:
picogreen
although this will also measure the template
another solution:
Run on the bioanalyser
this is pretty accurate
Hi I installed ImageJ, but i don't know how to qunatify my DNA.
I already found the function how to mark lanes, an how to plot the lanes, but for quantification i need the integrals - how can i get them?
thanks!
sven
hi,
you will find usefull informations about how to use ImageJ in this topic:
http://www.protocol-online.org/forums/inde...?showtopic=8201
Seb
I guess this is a repeat post as I'm sure I've already made a suggestion.
use the Bioanalyzer as asuggested above or use a nanodrop. These will both quantify small concentrations.
DO NOT use EtbR and ImageJ or NIH Image. EtBr does not intercalate uniformly into DNA and the UV lamp does not emit light in the same manner. Quantitation of bands using this method is not adequate given the number of better alternatives.
The best way is absolute quantitation using real-time RT-PCR but this is not the instant answer you seem to require.
hi,
sure, there is no more accurate solution than real-time PCR...
but EtBr staining, when correctly done, always gives a perfect standard curve line 
Seb
I know you said "fast", but sometimes it's quicker to just PCI/EtOH and measure on a spec than bother with implementing a new technique.
You cannot simply measure your PCR product on a spec because it most certainly contains unincorporated dNTPs and template, resulting in an overestimation of your concentration.
Running your PCR product against a set of mass standards is another way, and probably a bit faster/more accurate.
Hope that helps,
Hank
EtOH precipitation will remove the nucleotides; and the template will be in such small amounts to have a negligible effect on the absorbance measurement.
But How do i quantify my DNA?
No I am able to get the area below my intensity-peak, but i found out, that the intensity ist not linear to the amount of DNA. How can i solve that problem?
Do i have to calculate something with densitometric furmula?