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Plant DNA analysis by UV spectrophotometer indicates there is DNA in my sample,

electrophoresis plant DNA UV spectrophotometer DNA DNA Extraction

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#1 Charleen

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Posted 14 November 2013 - 08:44 AM

So I am comparing different kits for plant DNA extraction using corn callus and arabidopsis callus. I have used a Qiagen Kit, a Life Technologies (Invitrogen) kit and one other kit.  When running an agarose gel to get an idea of what (if any) size of DNA I have extracted from my plant cells only the Qiagen kit shows any DNA for either plant tissue.

I freeze the tissue in liquid nitrogen, grind with mortar and pestle, then do the extraction according to the kit instructions.

 

I have only been able to get enough DNA extracted by using the Qiagen Kit to be noticeable on my gels. The other 2 kits show nothing on the gel when staining with ethidium bromide.

 

So, I decided to do a UV spectrophotometer analysis of my DNA to see how pure it is. The DNA:protein ratios for one of the kits that shows no DNA on the gel is in the correct range, where it's supposed to be for purified double-stranded DNA (1.6-1.8).  However, I am not seeing anything on my gel!!

 

I have tried increasing the amount of DNA I load into my gels to see if that helps with visibility, and there is no change. Ladder shows up just fine, and the Qiagen kit DNA is somewhat faint, but it's there. The other 2 kits just show nothing at all.

 

Any ideas of why UV would be giving me absorbances indicating that there was relatively pure DNA in my sample, and yet nothing on the agarose gel? Not even any fluorescense in the well or anywhere in the lane for this kit.

 

Thanks for the help/advice.


Edited by Charleen, 14 November 2013 - 08:45 AM.


#2 bob1

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Posted 14 November 2013 - 12:02 PM

There are a number of things that absorb well in the UV range used to estimate DNA and RNA purity - these include other sugar based compounds and polyphenolics which are commonly found in DNA extracts of plants (usually coloured, so if you see a brown DNA pellet, then you probably have some of these).  Trust not in the 260:280 ratio as all this does is tell you if you might have pure DNA that doesn't contain protein - actually it was first used to tell if protein solutions were contaminated with DNA, it isn't all that sensitive for telling if DNA is contaminated with protein.  Other compounds may or may not interfere with the ratio.  The ratio is also dependent on the solvent you have used for your DNA - if you used water, the reading will be different to if you used TE or even just Tris.

 

Fluorescence on a gel is dependent on you loading enough DNA for you to be able to see it - it is a bit dependent on size of the DNA, but usually the lower limit is about 200 ng.







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