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DNA concentration from ultraspectrophotometer is not accurate


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6 replies to this topic

#1 MaggieRoara

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Posted 18 March 2009 - 01:32 AM

I did a maxi-prep using the kit from QIAGEN. I then did a 1000 times dilution of the DNA and did a reading using quartz cuvettes in my ultraspectrophotometer. The reading showed a good yeild of DNA, about 3.2ug/ul.
I then did the old fasioned way of gauging the DNA concentration by doing a serial dilution of the sample and loading the various dilutions in a gel with 10 ul of a DNA ladder. By comparing the DNA concentration of a reference band in the ladder with the various dilutions, my DNA content is actually possibly a forth of what the spectrophotometer told me it was.

Anybody got ideas as to why it is like that. The purity of my sample as measured by the spectrophotometer was okay.



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#2 HomeBrew

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Posted 18 March 2009 - 02:48 AM

My quick reply would be to read the title of your question as the answer to your question, as a general rule. There are many things that can throw your spec readings off...

#3 mdfenko

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Posted 18 March 2009 - 10:36 AM

do you blank the spec with the same solvent in which you dilute your dna?

do you dilute your dna with the same solvent that it is in?
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#4 perneseblue

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Posted 18 March 2009 - 10:57 AM

My quick reply would be to read the title of your question as the answer to your question, as a general rule. There are many things that can throw your spec readings off...



In addition, the spectrophotometer gives you the total DNA in your sample, including DNA fragments which individually are too low in concentration to be observed on a gel.
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#5 bob1

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Posted 19 March 2009 - 12:17 AM

As said before, EtBr fluorescence is not very sensitive, and will not pick up minor fragments that will be picked up by a spec.

The gel thing only works if you can load a serial dilution of the ladder, or even better spot (not run) serial dilutions of a known quantity of DNA onto a gel/sheet of plastic with dilutions of your sample side by side and then run a standard curve off the brightness of each spot.

Trying to estimate off one known point is completely useless.

#6 MaggieRoara

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Posted 19 March 2009 - 01:54 AM

As said before, EtBr fluorescence is not very sensitive, and will not pick up minor fragments that will be picked up by a spec.

The gel thing only works if you can load a serial dilution of the ladder, or even better spot (not run) serial dilutions of a known quantity of DNA onto a gel/sheet of plastic with dilutions of your sample side by side and then run a standard curve off the brightness of each spot.

Trying to estimate off one known point is completely useless.



How do I carry out your method? i gotta mix EtBr with the DNA then spot it onto a gel/ sheet of plastic?

#7 bob1

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Posted 19 March 2009 - 03:32 PM

As said before, EtBr fluorescence is not very sensitive, and will not pick up minor fragments that will be picked up by a spec.

The gel thing only works if you can load a serial dilution of the ladder, or even better spot (not run) serial dilutions of a known quantity of DNA onto a gel/sheet of plastic with dilutions of your sample side by side and then run a standard curve off the brightness of each spot.

Trying to estimate off one known point is completely useless.



How do I carry out your method? i gotta mix EtBr with the DNA then spot it onto a gel/ sheet of plastic?


Have a look in Sambrook, Fritsch and Maniatis, Molecular cloning, a laboratory manual. The full method is in there. Mix the DNA with an EtBr solution and then spot equal volumes onto a gel or sheet of plastic. Fluoresce on a UV source and take a photo, then do some densitometry on the spots. Be aware that saturation of the camera or photo will be reached at a relatively low level of DNA.




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