DNA A260/280 low

 Hi ,

Please I need help!! I start working with a nested PCR, my samples are bronchoalveolar lavage and after DNA extraction I got A260/280 very low - around 1.41, 1.56 and sometimes A260/280 very high - around 7. I know a good ratio of ~ 1.8 is pure DNA. How can I interpret these results? Very low A260/280 seems that I don't have DNA? And higher results? I didn't get any band in my PCR with this samples.

thanks
Carol12

Hi Carol,

When it comes to 260/280 ratios, anything outside ~1.8 is simply not good. In my experience sometimes you can get a low ratio (as low as 1.6) and still have DNA that can be used for PCR, but for the most part you should try to get DNA that has a ~1.8-2.0 ratio. 

If you are extracting your DNA using a home-made protocol, my recommendation is to purify your DNA using a commercial kit. A kit is of course more expensive, but kits work very well and you can easily determine if the problem with your DNA is coming from your protocol or not. 

Another thing you can do is run your DNA on an agarose gel. If you have good DNA you will detect a very high molecular weight band at the top of your gel (right below the well of the gel). If on the other hand your DNA is not good, you will likely get a smear, or maybe the DNA will remain stuck in the well (if it has a lot of impurities). 

Good luck

 Thank you so much Ivan for your reply!!

Actually I did the extractions with Qiagen kit and after  these bad  results I did extraction with chloroform - better results. My samples are frozen with a viral transport medium, maybe it interferes in my extraction.

I will run my DNAs in agarose gel as you suggested me :)

I have more than 100 samples, I have to check  the DNA in all of them before my PCR,  is it?

Thanks again!!!
Carol

Hi Carol,

I just wanted to add a couple of suggestions to what Ivan has already posted. A low value for 260/280 could indicate impurities. However, a value of 7.1 could also indicate that your spectrophotometer may not be sensitive enough to measure the concentration you are working with. If you are using a regular spect to meaure 5 or 10 ng concentrations, this could be the case. This could also be related to why you didn't see amplification.

After chloroform extraction, are you also using ethanol precipitation afterward? One method taught to me years ago was to concentrate PCR or plasmid using glycogen as a carrier. Typically we use 1.5 ul glycogen (Roche), 80 ul of 95 percent ethanol for a 20 ul sample. This is followed by a 200 ul wash with 70 percent ethanol.

I hope this helps.

DougB

Hi Carol,

I wouldn't check all your samples, just a few to make sure they look ok. In theory all the samples that were purified the same way should show similar results in a gel. 

I agree with the suggestions provided by DougB. Having too much DNA is rarely a problem, mainly because it is not that common to use too much material when extracting DNA, but having too little DNA can certainly cause a problem, since spectrophotometers require a minimum amount of DNA to give reliable results. 

Cheers

 Thanks DougB!! I'm using a nano spectophotometer.

I'm doing a qualitative PCR. A good amount of DNA for it is ~ 30 ng/uL, isn't it? Maybe I didn't get bands because my DNAs have ~2 ng/uL. What do you think?

thanks :)
Carol

Hi Carol,

In my experience qPCR can detect ~2ng/uL, but only in some cases when the assay you are using is very sensitive. A more average sensitivity for a qPCR assay is 10 ng/uL, so using 30 ng/uL would be a good starting point. All this assumes that you are using at least 1 uL of DNA per reaction.

 Thanks Ivan! You're helping me a lot!

I'll try to obtain more DNA to use in my qualitative PCR (Nested).

Hi Carol, I will also have to give credit to Ivan. My experience in qPCR is somewhat limited. However, I will say that it is possible to amplify from 2ng of DNA for well optimized PCR. ABI AmpliSTR is a good example. The kit is capable of 2 ng sensitivity amplifying with 10 multiplexed markers. But I would imagine it required a team of scientists to develop the protocol. Good luck. Please let us know about your successes on the study or if we can provide any more help. DougB

 Hi Ivan,

I ran my DNA on agarose gel. Some samples with ~ 2 ng/uL I could see a weak band .Even I saw the DNA in gel this amount  isn't good for my qualitative PCR, is it? I'm trying to repeat some  positives samples that were analysed before me but I didn't get band, I don't know how much DNA was used before.

Have a great weekend!!
Carol

Hi Carol,

If you saw a weak band in an agarose gel, then very likely you have more than 2 ng/uL. As far as I can tell agarose gels can only detect ~40 ng or more DNA, although this depends highly on the method of detection. 

Running your DNA in an agarose gel is more to make sure that your DNA is not degraded than for quantification purposes. The fact that you got a single tight band suggests it is good quality DNA. 

 Hi Ivan,

My assay is getting better :) my PCR is working very well.

But I have more one question...sometimes I have ~ 1.8 ng/uL of DNA and it appears in agarose gel. But today I had ~ 18 and 9 ng/uL and they didn't appear in agarose. I don't have DNA in these samples? How the nanodrop did the measure? If I do a PCR  it won't work?

Thanks a lot,
Carol 

Hi Carol,

I am glad the assay is getting better. 

I really do not understand why you cannot see DNA in an agarose gel that is ten times more concentrated than DNA you can see in an agarose gel that is a tenth of the concentration. My recommendation in this regard is to contact the people the sell the Nanodrop and ask them to explain why this is happening. 

Likely the PCR will not work if you cannot see the DNA in the agarose gel, but it may be worth trying anyway.

 Ivan, I'm repeating the PCR, these samples were analysed before me. Could the DNA is not good because has been frozen/thaw before me?

thanks

Hi Carol,

Are you using a standard with a known concentration when you set up the Nanodrop? We have an in house standard that we measure before measuring unknown samples. Large fluctuations could indicate a problem with the Nanodrop. I would also recommend a couple of water rinses before measuring a sample with concentration below 20 ng per ul in case there is residual DNA from a previous sample.

Let me know if this helps.

DougB