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DNA A260/280 low

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Ivan
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Posted By Ivan
on 5/17/2012 6:28 AM   
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Hi Carol,

There a number of explanations for what is going on, but most likely there is an issue with the reading you are getting. As DougB suggested you should make sure the readings are made with a clean instrument.

As for the DNA, you should never freeze and thaw DNA. Freezeing and thawing DNA degrades it. Whether that caused the changes in the readings it is possible. 




Ivan Delgado Orlic Carlsbad, CA



Carol12
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Posted By Carol12
on 5/20/2012 16:31 PM   
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 Hi DougB,

I don't have a standard with a known concentration when I set up the Nanodrop. I think nobody in the lab has a standard but I'll ask my collegues.
I did a DNA extraction with QIAamp kit and the same sample with chloroform, with chloroform I had more than 100 ng/uL but I ran in an agarose gel and no band! Some samples have bands in the gel but others no.

Thanks for your help :)
Carol




DougB
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Posted By DougB
on 5/21/2012 7:41 AM   
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Hi Carol, A sample showing 100 ng per ul but not showing on agarose is a little strange. The Nanodrop scan will provide the concentration of the sample and whether certain salt contaminants are present. However, it will read the same whether DNA is supercoiled, nicked or in small pieces. For example, we use the Nanodrop to measure 18 base primers as well as plamid DNA. Many years ago we saw cases where plasmid preps were purified using resin based columns. Resin was sneaking through the membrane into the final sample. Over time, the resin re-absorbed the DNA. But I haven't seen this problem for a long while. I am thinking possible degradation. Partially degraded DNA could still read on the Nanodrop then electrophorese completely through the gel. Are you seeing even a smear on the gel? Any thoughts Ivan? DougB



Carol12
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Posted By Carol12
on 5/21/2012 8:02 AM   
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 Hi DougB, I saw no smear, nothing!! But some samples with 1.8 ng/uL I saw a weak band. For me it seems there is no DNA in the sample. The samples were frozen/thaw before me but I think the DNA should  be viable.

Thanks :)
Carol



Ivan
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Posted By Ivan
on 5/21/2012 8:18 AM   
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I agree with DougB that you can get a reading in a Nanodrop, suggesting you have DNA, and then get virtually nothing on a gel, mainly because the DNA is degraded. Having said that, if you have a concentrated sample, even if it is mostly degraded, you should see at least a faint blob of nucleic acid towards the bottom of the gel (around 50 base pairs in size), which is typical of fully degraded DNA. Of course this assumes you are not running your DNA dyes off the gel

Based on the posts in this thread my best guess is that you simply do not have DNA. Wether this is because the extraction is not working, your starting material is too limiting/degraded, or something else is an open question. My suggestion at this point would be to obtain a fresh sample, for example some liver, and go through the whole process and see if you can get a good amount of DNA that is easily detected by the Nanodrop and seen on a gel. If you cannot get that to work with a liver sample then there is a problem with your DNA extracion procedure. If on the other hand you get a nice amount of DNA and you can detect it in a gel, then the problem is likely with your bronchoalveolar lavage samples. 


Ivan Delgado Orlic Carlsbad, CA



Carol12
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Posted By Carol12
on 5/21/2012 17:14 PM   
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 Ivan, I'm running 5 ul of the DNA with 1 ul of a loading buffer 120V 20 min, is it  wrong? thanks



Ivan
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Posted By Ivan
on 5/21/2012 17:29 PM   
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Assuming you are using a 6x loading buffer, then that sounds fine. As for the gel conditions, as long as you are running a size marker and the marker bands run as expected, then any gel conditions you are using should be fine. 


Ivan Delgado Orlic Carlsbad, CA



Carol12
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Posted By Carol12
on 5/23/2012 14:43 PM   
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 Dear Ivan,

More one question...I sent my pcr product  for sequencing and the foward primer is good but the reverse is not working. Can I work with just one primer working or I have to redesign the reverse primer? The PCR is working, the positive control is good.

thanks
Carol



Ivan
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Posted By Ivan
on 5/23/2012 15:47 PM   
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Hi Carol,

It is not uncommon for the reverse primer to fail a DNA sequencing reaction, in particular when you are sequencing a gene that has a typical 3' end. Whether you should get the reverse sequencing reaction to work or not depends on your needs. If all you need is to confirm that you are amplifying the corrrect piece of DNA, then all you need is the DNA sequence obtained from the forward primer. If on the other hand you need to clone your PCR product and use it for aplications such as protein synthesis, then I would make sure that both ends are sequenced.


Ivan Delgado Orlic Carlsbad, CA



hcheung
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Posted By hcheung
on 6/26/2012 5:23 AM   
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According to the GE handbook "Nucleic Acid Sample Preparation for Downstream Analyses", p. 50, A260/280 <1.7 Proteins or chaotropes may be present, while A260/280 >1.9 RNA may be present. They provide further advice on how to remedy including:

- do not overload the purification system
- treat with protease or perform a phenol extraction to remove protein contaminants

To get the handbook:
http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_19146/28962400



Carol12
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Posted By Carol12
on 6/26/2012 6:17 AM   
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 Thank you very much hcheung :) your post help me a lot!!!

Thanks everybody!!! I really appreciate your effort to help me :)

Best wishes for everyone!!!
Carol12



Anna E
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Posted By Anna E
on 6/30/2012 12:18 PM   
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Hi Carol.
I hope your experiments are working out now. I just want to share some DNA quantification and quality analysis perspectives with you. I have experienced the same problems as you many times and up until a couple of years ago I had great fate in using the Nanodrop for quantification and even for quality analysis. However, as I was exposed to problems related to extracting pure DNA from autotrophic organisms with complicated cell walls that contaminated the DNA and resulted in PCR inhibition and unreliable qPCR results, I learned that the Nanodrop only gives you very rough estimates - especially when you work with low concentrations of nucleic acids. Also, many cleanup kits contain polysaccharides (a PCR inhibitor) in the actual cleanup matrix.  It is unfortunate but luckily there are ways to get around this.

When working with the Nanodrop it is good to remember that RNA (260 nm) and DNA (280 nm) absorbance overlap to a high extent and unless you have treated your sample with RNase some of the reported DNA concentration is actually RNA. Also, if the 260/230 value is too low you can not rely on the 260/280 value at all. I would skip using the Nanodrop for low concentration samples and use the Qbit fluorometer. It gives VERY accurate results. For quality and integrity analysis of DNA (and RNA) use an Agilent bioanalyser. From there you can verify your method improvements.   Let me know if you want more information on the methods I am suggesting. They work!

 

Good luck with your PCRs!

 

Anna




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