How to get a good standard DNA quantification curve - (Apr/13/2005 )

Hi!

I'm having trouble making standard curves for DNA quantification. I can get a nice linear curve, but when I try to repeat the same experiment, even the blank value has changed...

I'm using Hoechst 33258 (fluorescence) according to the manuals, but with an incubation time about 6 min. This is since I observed that the fluorescence intensity increased with time (why?).

How do you know when you get "the right curve"? Is it supposed to be this variations?


*troubled*

Sorry I've not used fluoresence, but I was wondering if you could use UV?

Hi
sarah 123 meant that you could do a spectral analysis by uv ranging 220 to 320 nm...

Ok, thanks, I suspected that... One reason why I don't use absorbance is because our spectrophotometer is a piece of crap While our fluorometer is a nice new ting..
Besides, I've heard that DNA measurements using spectrometry can be influenced by contaminating proteins?

have you tried using SYBR green dye? Picogreen from Molecular Probes gives a very consistent standard curve. Very sensitive and has a large linear range.

Nick

hi

Hi there..
Did you ever get chance to use Picogreen assay? If you have how were the results? I am having hard time getting a meaningful results from my microplate reader.... any suggestions would be highly appreciated..

Regards,

Letmetellusome

Hi!

I'm having trouble making standardcurves for DNA quantification. I can get a nice linear curve, but when I try to repeat the same experiment, even the blank value has changed...

I'm using Hoechst 33258 (fluorescence) according to the manuals, but with an incubation time about 6 min. This is since I observed that the fluorescence intensity increased with time (why?).

How do you know when you get "the right curve"? Is it supposed to be this variations?


*troubled*

Hi!

I now only use Picogreen, and it seems to be working quite good. Picogreen gives, in my opinion, rather stable values (as determined over a time series), and I kind of "trust" the results.

In essence I followed this protocol: http://plantpath.wisc.edu/~amyc/Protocols/...ntification.htm
but used a longer incubation time (6 min) since my time series indicated that at this point the values were much more stable.

When I started I thought that making a standard curve was nothing. It turned out to be rather tricky. I wouldn't exclude the possibility of hardware or software problem. We had a lot of those, though our machine is new!
Are your microplate reader aligned to the plates you use?

Hi
I am using a Victor, 485/520 nm and the PicoGreen for quantification.

I am using the PG for WGA quant. and low conc. gDNA quant.

My problem is, like others in this forum, that the curve equation differs from plate to plate, ranging from 6 to 14x, giving me a hard time concluding anything about the concentration of the samples..

Is anyone aware of a way of solving this? Should one make THE curve and then let it be the golden standard??

Hope somebody can help me with this...