Quantification of DNA

[peacefulspring]

Hi,

Can anyone kindly tell me how to quantify DNA other than using spectrophotometer/ gel electrophoresis. And how could we measure DNA using only 1microlitre?

 

Thanks.

[CharonY]

Well you could do it with a nanodrop (only needs 1µl), but is also a spectralphotometric method. Alternatives are a fluorescence based assay with subsequent measurement with a fluorometer (e.g. picogreen). You could also make isotope dilution measurements with a MS, but that is kind of overkill.

[ecoli]

nanodrop is your friend.

[GDG]

There's always quantitative PCR.

[CharonY]

Well, yeah, but you would need identical DNA as a reference standard to be able to measure abundance.

[peacefulspring]

But if I use Ethidium Bromide Fluorescence for the amplifiable DNA from maize (For the detection of GM maize experiement), is it alright too?

[CharonY]

It is possible but :

- it is overall rather inaccurate

- you need a reference standard in the same run with about the same length of the fragment(s) you want to quantify (as the incorporation of EtBr is length dependent)

[peacefulspring]

Thus, if I choose to use Fluorometric Quantitation for my maize's DNA, would it be better?

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besides that, usually for negative control in detection for maize dna for instance, is the content of the negative control containing

1) only distilled water

2) everything (e.g. primer,polymerase & stuffs) except for DNA

[CharonY]

Technically yes, as methods as e.g. picrogreen have a higher level of linearity (around three orders of magnitude). You still need to make a standard curve, though (is true for every quantification technique, actually). The length dependency is also a bit lower than for EtBr.

 

And 2 is the way to go. For controls you should have precisely the same parameters except the one you are testing.

[peacefulspring]

besides that, what would be the optimum voltage for 2% agarose gel and the duration for it? In addition, is the pore size of 2% agarose bigger than 1% agarose?

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in addition to the previous post, the amplified products ranges form 100-600bp in size.. can anymore rly asap cos it's urgent!thanks..

[lucaspa]

besides that, what would be the optimum voltage for 2% agarose gel and the duration for it? In addition, is the pore size of 2% agarose bigger than 1% agarose?

 

The pore size of a 2% agarose gel would be smaller than a 1% agarose gel. More agarose, denser the agarose molecules and the smaller the pores between them.

 

No matter the voltage, you can track the migration of DNA thru the gel with the dyes bromphenol blue and xylene cyanol. http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html

 

However, "As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel). "

 

I would suggest you do 5 volts per cm.

[CharonY]

If you want to have good resolution down to 100 bp you should definitely use 1.8-2% gels. Regarding voltages, it also depends on buffer capacity. As a rule of thumb, lower voltages generally yield better resolution. However the trick is also to stop the gel at the right time, depending on where you expect the majority of bands.

Edit: many minigels have a size of 8-7 cm. But running at 5V/cm (or 40-35 V in total) is not really necessary. Usually anything from 50-80 V generally works pretty well.

Edited May 12, 2009 by CharonY

[peacefulspring]

I've another question regarding DNA extraction from maize.

So can anyone kindly teach me how to make fresh kernels to powder form? Is it by using liquid nitrogen so that the corn powder that we obtained would be to be extracted? And after which, can we simply use food mixer/ pestle/ mortar to 'grind' it to the powder form that we want?

Thanks.