Genomic DNA clean-up is a technique that is very common but still causes many people to suffer from separation anxiety. Here’s a look at some tips and tricks to improve your DNA clean-up and avoid the loss of precious samples.
Why is my DNA “dirty”?
Here’s the scene: You’ve collected a set of samples, they could be plants, seeds, stool, gut material, soil, water, FFPE or anything that is known to contain PCR inhibitors. You isolate DNA using your typical method and the DNA fails to amplify in PCR. You need every last molecule for whole genome shotgun sequencing…. what do you do?
Secondary clean-up of DNA
The PowerClean DNA Clean-up Kit provides a quick, easy and reliable secondary clean-up method to purify previously isolated genomic DNA from any source. The kit contains MO BIO’s patented Inhibitor Removal Technology (IRT), a proven method of removing PCR inhibitors including humic acids, polysaccharides, polyphenolics, lipids, heme and more.
Going back to the sad story of your precious samples that failed to amplify… After learning about PowerClean, You run the samples through the kit to isolate clean DNA. But wait… the spec readings have changed! Before clean-up it said you have 300 ng/ml of DNA and now it says you have 30 ng/ml. It’s your worst nightmare. Your DNA has been lost!
Don’t worry, we’re here to tell you that your DNA is safe and sound – and clean. There’s some confusion about how to tell what you had before and after clean up using standard quantification lab techniques.
PCR inhibitors influence DNA quantification
Previously, we explained how PCR inhibitors wreak havoc on UV260 wavelength readings and inflate DNA yields. This is important because many of the organic inhibitors found in plants, seeds, stool, gut material, soil, water and FFPE tissues co-absorb at the 260 wavelength and interfere with accuracy in DNA yield readings. Degraded RNA, which will be co-isolated with CTAB or phenol:chloroform methods, also interferes with absorbance readings. Silica spin filter kits that do not have a method for inhibitor removal and use very strong binding salts that do not discriminate between DNA and RNA binding are also problematic. All of these factors are going to cause problems when interpreting your DNA yield.
The good news is, once these interferences are removed, the new absorbance readings represent the true DNA yields. Let’s look at a real life example of how this will look using standard laboratory techniques; the NanoDrop and agarose gel electrophoresis. Beginning with the NanoDrop (Figure 1), we see the results of the same soil extracted with and without Inhibitor Removal Technology. The first thing everyone looks at when evaluating their DNA prep is the ng/ml yield reading. This is important but it is only a small part of the story.
Figure 1. NanoDrop readings for samples with (No IRT) and without (IRT) PCR inhibitors.
The 260/230 ratios in Figure 1 tell us that there is a problem with this DNA. In the samples with inhibitors (No IRT), the readings are below 1.0, indicating a high level of impurities. We can get another key piece of information from this data; the 340 reading. The samples isolated with no IRT have an elevated 340 value compared with the pure samples isolated with IRT. They are almost 10X higher. Humic acids will optimally absorb at wavelength 320 so the high absorbance at 340 reflects the carry-over of humic acids in the final DNA. The 260/230 ratio, combined with the high 230 absorbance, tells us this DNA is not clean due to the presence of organic compounds.
NanoDrop also provides graphical representation of the absorbance data across all of the wavelengths (Figure 2). We clearly see a high level of interference across all of the wavelengths being measured. It starts high and stays amplified. The 260 reading is caught in middle of this inhibitor-fest and consequently is way above where it should be.
Figure 2. NanoDrop absorbance data for samples isolated with and without IRT.
However, we know that it is still difficult for some people to believe that this is not all DNA. In this case, best way to confirm your results is with an agarose gel picture. The gel picture has information that is not visible on the NanoDrop; integrity of the DNA and a visual representation of the yields (Figure 3). Although the NanoDrop indicates that the No IRT samples are more than double the yields of the IRT samples, the gel picture shows us that the yields of the No IRT samples are actually lower. The contaminants make analysis confusing. This is why we always recommend checking your DNA with two methods – NanoDrop along with a gel picture or PicoGreen reading.
Figure 3. Gel analysis of samples isolated with and without IRT.
So what happens when you clean dirty DNA samples up using the PowerClean Kit? You remove these interfering compounds from the DNA and, as a result, you see a drop in the yield reading. In this case it would be more than 50%. However, this decrease in yield is not due to the loss of high molecular weight DNA. It is loss of the compounds you don’t want in your prep. It is now pure DNA.
RNA contamination can inflate DNA yields
Depending on the method used, RNA will co-extract with the DNA. It may not be intact, but if it is there it will absorb UV. Any method using phenol or chloroform to extract nucleic acids will co-isolate the RNA, and methods using strong binding salts and ethanol in equal volume will also result in RNA co-isolation. Because the NanoDrop cannot differentiate between DNA and RNA, this is where a second method such as PicoGreen comes in handy, because it measures dsDNA only. An agarose gel picture is also helpful because the RNA smear can be visualized.
In this article, The Difference between the NanoDrop and fluorescent dye for quantification of DNA, we show how much RNA can impact your yield readings.
We can demonstrate this by examining plasmid DNA purified from 4 ml of overnight LB E.coli culture using a popular plasmid prep kit on an agarose gel (Figure 4). Here, we can very easily see the degraded RNA smear at the bottom of the agarose gel. The average NanoDrop reading for these 3 samples was 81 ng/ml, while the corresponding PicoGreen reading averaged just 16 ng/ml. RNA absorbance can impact readings by as much 70% for some samples. This amount of inaccuracy in your DNA sample can lead to pretty big errors later on.
Figure 4. Agarose gel analysis of plasmid DNA isolated from 4 ml of LB E.coli culture
Accurate yields and clean DNA
So the take home message is this: examine DNA samples before clean up and after clean up on an agarose gel. Take a look at the intensity (yield) and integrity (size range of the DNA) and compare with your second method (NanoDrop or Picogreen) to check for the presence of RNA or inhibitors.
You can save yourself anxiety and time if you do a simple check to see what you are starting with. Remember, the PowerCleanâ Kit can only give back what you put in. You want clean DNA. But knowing how much DNA you had to begin with will calm your nerves and give you the assurance you need about the outcome.
Want to try it for yourself? Request a free sample of the PowerClean DNA Clean-up Kit today!
Integrated DNA Technologies (IDT), the world leader in oligonucleotide synthesis, will be presenting at the upcoming qPCR 2010 International Symposium, 7 – 9 April, in Vienna. Dr Mark Behlke, IDT’s Chief Scientific Officer will talk about, ‘Hydrolysis probes with improved quenching and performance’, which addresses the requirement for efficient quenching to optimise probe performance. The […]