If you’ve figured out how to extract high-quality DNA from an elephant, chances are that without too much trouble, you’d be able to do the same from a moose, a mouse or even a meerkat. However, if you’ve figured out how to extract DNA from an Arabidopsis plant, well that might be about all you’ve figured out. That’s because plants have developed something akin to chemical warfare in order to survive a variety of climactic extremes, pathogens, and predators, without the luxury of being mobile. As a result, plants harbor an enormous variety of organic compounds, some with antifungal and antimicrobial properties and some which make them taste bad to herbivores. Other structures are complex networks of polymers that store water and nutrients for both feast and famine.

It’s all good and well for the plants, but many of these substances muck up DNA extractions....

The DNase step is one of the most common causes of degradation or loss of the RNA during your extraction. DNase digestion is frequently performed on the spin column and although this can be a great way to save time on the post extraction processing, it is not an efficient method for samples with large amounts of DNA (for example, spleen, thymus, and even some soils). In these cases, DNase digestion in solution is necessary.

The typical protocols for DNase involve inactivation of the enzyme using EDTA and heat. Both of these things can cause problems in RT-PCR. EDTA can inhibit the RT-PCR enzymes and heating the RNA can cause a reduction in integrity. 

As many of you know, a good percentage of our customers are doing microbiome research. Microbiomes, those localized communities of microorganisms that exist symbiotically with their immediate environment, can be found virtually anywhere; inside human colons, around plant roots, inside coral reefs and even within ant colonies. These little microbial microcosms are a hot topic right now. Recent studies have implicated their role for the wellbeing of people, animals, plants and entire oceans. Variations in microbial numbers and diversity within animals have shown critical involvement in auto-immune disease, obesity, acne, tooth decay, pregnancy and even brain chemistry. Plant studies have shown that symbiotic organisms impart resistance to drought, increase nutrient absorption and prevent attack by pathogens.

QIAGEN is knee-deep in questions about DNA and RNA isolation from stool! One of the most frequently asked questions is how to store fecal samples for shipping and then storage for further processing. We consulted a 2010 paper written by Dr. Rob Knight’s lab¹. Dr. Knight is an expert in the microbiome field working with human gut/stool samples.

QIAGEN is cleaning up...DNA and RNA that is. With the addition of the DNeasy and RNeasy PowerClean Pro Cleanup Kits to our portfolio, we thought it would be a great time to discuss how cleanup kits work and when you'd want to use one. Clean-up kits take dirty nucleic acids and remove contaminants that could interfere with your downstream business. Enzyme-dependent applications like restriction digests, ligations, PCR amplification and sequencing all require squeaky clean DNA and RNA in order to get the best (or sometimes any!) results. 

I recently met Ermanno Federici in Milan, Italy while attending Bacterial Genetics and Ecology (BAGECO 2015).  He described to me a "Wall of MO BIO" in his lab, and sure enough, true story!!  Here is a little bit about what happens in his lab... thanks for sharing!!

"Our main interests is microbial ecology and, in particular, the use of culture-independent molecular methods (qPCR, PCR-DGGE, and Illumina NGS, …) to investigate environmental microbial communities and their role in ecological, biotechnological and pathological processes."

When the BiOstic® Bacteremia DNA Isolation kit was originally designed, it was optimized for the extraction of bacterial DNA from infected blood. However, it has turned out to be useful for so much more. Bacteremia is the presence of bacteria in the blood and under normal circumstances blood should be sterile, but with the insertion of a catheter into the body or through an open wound, bacteria can gain entry and take hold. Even a small numbers of bacteria can be a real problem...

One of the most efficient ways to extract nucleic acids from a sample is by smashing it against a hard surface repeatedly under high speed until cell walls and membranes crush from the pressure and release their internal contents. In other words: bead beating.

Bead beating is a great way to do what enzymes take hours to accomplish and sometimes never fully succeed in, which is cell lysis to release DNA or RNA for isolation. While enzymes can be successful for DNA isolation from a limited number of sample types, results are achieved a lot faster if you break down the matrix first. And RNA cannot be isolated in a timely fashion without the use of some kind of mechanical maceration.

The questions inevitably arise though, how hard do I need to beat to lyse my sample and how do I know what bead type to use?  The answers depend on a great number of variables, so to avoid beating your head against the wall to sort through them all, I have written this two-part blog series offering advice on the methods that we have used at MO BIO and found to work best for us.

Formalin-Fixed, Paraffin-Embedded (FFPE) tissues, the most common tissue preparation method for archiving bio-specimens, can be a valuable resource for genetic studies. The fixation process makes it possible for samples to be stored for years at room temperature, for analysis even decades later.

Chemical fixatives like formalin (a mixture of formaldehyde and methanol) preserve the structural integrity and morphology of the tissue by cross-linking neighboring amino groups between proteins. This traps other molecules like carbohydrates, lipids and nucleic acids in place.

While the fixation process preserves the structural integrity of the cells, it also denatures proteins and causes the degradation of RNA and DNA. The longer the fixation process and the age of the tissue the more damaged the nucleic acids.  As a result, the size of the nucleic acid fragments is generally small, in the 100-500 bp range. All this makes the extraction, amplification and analysis of nucleic acids a bit tricky. However, using appropriate protocols and kits it’s still possible and highly worth the effort.

Blood contains a mixture of plasma, red and white cells and platelets.  It is a unique beast among sample types, because while the quantity of nucleic acids in blood is copious, this genetic gold mine bathes inside a complex soup of cellular debris and protein. These contaminants can interfere with downstream PCR and sequencing.  It is the hemoglobin in particular, within the red cells, which causes major issues in DNA/RNA contamination and PCR inhibition.

Dear MO BIO,

I am using the RNA Power Soil Total RNA Isolation kit with some high saline soil.   I was not able to extract any RNA but was able to extract DNA with the kit.  What's going on?

Thao

One of the top five technical concerns we get at MO BIO relates to low yields of DNA.   Whether it be sediment, swabs, waste water or sludge, at some point we've probably heard that you didn't get enough DNA out of it.  After all, there is no such a thing as getting too much DNA.   Nope, haven’t heard that grievance even once!

When someone tells us they didn’t get enough DNA, we rule out the obvious offenders. It could be that your lab-mate left your samples out all night at room temperature and didn’t tell you. It might also be that you’ve made an error in the way you’ve interpreted or conducted a protocol or perhaps you’re using an inappropriate kit. The list goes on and on.  However, the answer could be a lot simpler.  Low DNA yields (defined here as those that can't be easily measured with an absorbance spectrophotometer, for example, a Nanodrop) may not indicate that something went wrong at all.   It might just be that there wasn't a lot of DNA to begin with.  This is very common with low biomass samples like swabs, filters or sediment.

Considering a move to high throughput DNA/RNA isolation? Many of our customers, who’ve been using MO BIO single prep kits for some time, now want to scale up.  We’ve been getting a lot of questions as to what equipment is required to make the switch.  Scaling up usually means moving sample preps over from 2 ml tubes to standard 96 well microplates. While many customers have experience with multichannel pipetting for these plates, many have never done any bead beating in 96 well format.

Most MO BIO kits take advantage of bead beating to assist in cell lysis because it is an exceptionally good method for isolating gram+/- and spore and oocyst DNA & RNA.  But bead beating, while easily done on a vortex in 2 ml bead tubes, requires much beefier equipment for 96 well plates.

Three years ago we published a tech article in which we went into detail on using swabs with MO BIO DNA Isolation kits.   Recommendations depend both on levels of PCR inhibitors and whether one wants microbial or eukaryotic DNA.  The article included details on how to transfer swab samples to bead tubes and collection tubes for use in each kit.  Swabs continue to be one of the fastest and easiest methods for sample collection and remain a popular subject for tech support questions.

Since the previous swab article, we’ve developed two new kits that we recommend for swabs, one for RNA Isolation and the other, for high through-put rapid mechanical lysis of microbial cells on swabs from low biomass/low inhibitor samples.

Most of the time choosing a DNA Extraction kit is pretty straight-forward.   If you want to isolate DNA from lung tissue, then use a tissue DNA isolation kit.   If you want DNA from microbial culture, then use a microbial DNA isolation kit.    But what if you want microbial DNA from the lung tissue?  Do you choose the tissue kit or the microbial kit?  And what if you want animal tissue DNA from a soil sample?  Should you use a tissue or a soil DNA isolation kit?  It gets a little tricky.  These kinds of mixed up situations make up a good fraction of our technical support questions.   We have worked out protocols for many of these scenarios.   Below we discuss three of the most common mixed-up circumstances and give suggestions as to which kit and protocol will work best for them.

A few years ago the folks here at MO BIO technical support were contacted by a customer from UC Berkeley who was trying to extract microbial DNA from Western Blue Bird fecal sacs. She’d tried several sample kits of PowerSoil. Yet, despite her best efforts, she could not get any measurable DNA from this particular sample type. We knew that the DNA had to be in there. After all, fecal samples are chocked full of microbial DNA. We went down our normal list of troubleshooting suggestions. The sample was collected properly, stored properly and she was following the normal protocol. But still no luck! So what the heck was going on? We  had extracted DNA from many different types of fecal samples in the past without any difficulty. In efforts to figure out the answer, we suggested that she send us a few of the bird fecal samples so we could do some experimentation.

Whether you don't know which DNA isolation kit to buy or you just have a protocol question, MO BIO technical support it here to help. More often than not, we've heard a similar question before and we can answer you pretty quickly. Occasionally, however, it takes some brain wracking. It might be that you've managed to throw something unique our way. But usually, it's because we don't have enough information to go on. In that case, we will write or call you back, ask for more specifics and then (depending on the answers) we might ask some more. While we all enjoy the back and forth banter, we know you'd really like to get back to your research as quickly as possible. So we thought maybe we should come up with some tips for you to make the process more stream-lined. We were able to get it down to five key elements. This list is not comprehensive. But, we think if you can give us this information right off the bat, we'll be able to get you back on track fast.

Dedicated bead beating instruments are costly. For a device that can hold more than just a couple of bead tubes, prices quickly rise into the thousands of dollars. Fortunately, a less expensive alternative, the humble and multipurpose vortex, can do the trick nicely for most bead beating applications. For optimal results, we recommend that you use a vortex adapter, a detachable horizontal tube holder, for efficient and consistent homogenization of your samples.  MO BIO manufactures such adapters in a variety of tube sizes from 2 ml up to 50 ml.

While the majority of our customers use these adapters, sometimes their MacGyver instincts take over and they will rig up something of their own.  And while they have come up with some pretty clever and unique constructions, the results are not always favorable. Though it may seem like a remarkably simple device, much testing and optimization has gone into the design of our vortex adapters. From the orientation of the tube holders to the number of tube holders on each platform, each one has been designed to work best for a particular type of bead tube. 

We speak with many scientists who work with filtered water for isolating microbial DNA and RNA. Water samples can be difficult because of their typically low biomass (depending on the water source) and because these samples are often from precious and unique sources.

Why is molecular research on microbes in water difficult?

For some people, getting back to the original source of water may not be possible for months or even years. For example, we talk to scientists collecting samples at hydrothermal vents in the middle of the ocean, in the Antarctic, and in the Baltic Sea.  For some researchers, water samples may have been collected after a certain event, such as a flood or heavy rain and so the conditions of the water will not be the same in a week or even after a day. They need to get answers from every sample collected and they need it to accurately reflect the current microbial content.

This week we want to discuss a technique that is very common but still causes many people to suffer from separation anxiety.  What could that possibly be? It is cleaning up dirty genomic DNA.

Here's the scene: You have a precious soil sample collected from the roots of an ancient never-before seen orchid located on a remote island in the middle of the South Pacific. You isolated the DNA from microbes in this soil using a method other than the PowerSoil Kit. It's still dirty and won't amplify in PCR so it needs to be cleaned of humic acids and other humic substances. You need every last molecule for whole genome shotgun sequencing.... what do you do?

We know many of our customers like to be selective about their RNA. That's because, most of our RNA technical questions involve a desire to retain or exclude certain varieties of RNA. It’s not always possible to get what you want;  but sometimes by making slight adjustments to the extraction protocol, it is possible to get what you need. In fact, in a previous MO BIO blog article [microRNA from Fresh Tissue and FFPE Samples using MO BIO Kits with Modified Protocols] we discussed how to bring in very small sized RNA when using our tissue extraction kits. 

Since many of our customers are now turning their efforts towards extracting RNA from more difficult samples (AKA dirtier), we figured it would be a good time to talk about some of our newer RNA kits: PowerMicrobiome RNA, PowerPlant RNA, PowerWater RNA and PowerBiofilm RNA. All four of these RNA Isolation kits use the same combination of two solutions to bind the RNA in your cell lysate to a silica spin column. It's the ratio of these two binding solutions that determines the size of the RNA that will bind and subsequently the size of the RNA you extract. Everything smaller than the cutoff point will wash off the column.  Everything above the cutoff will stay on the column and will come out in your elution. You can alter the binding solutions to change the cutoff point but you’ll always lose everything smaller than the cutoff.

Microbes outnumber the human cells in our bodies by about 10 to 1. So wouldn't you like to know which ones are hitching a ride? Well now, thanks to a new open-access project known as “American Gut” nearly anyone can find out about their own personal community of microbes.  In association with the Human Food Project, researchers at the University of Colorado Boulder along with nearly thirty researchers at other institutions around the world, are on a mission to understand the interaction between diet, lifestyle and our microbes. The project builds upon previous efforts, including the five-year, $173-million NIH-funded Human Microbiome Project, to characterize the microorganisms living in and on our bodies. However, unlike other projects that worked with just a few hundred hand-selected test subjects, the American Gut project allows anyone in the public to get involved.  In fact the hope is that tens of thousands of people will do so.

The project is not just an exercise in curiosity.

Today I wanted to talk about a method that you’ve been doing forever in your science career. Something so basic, so easy, that I bet you don’t even have to think about it. I bet you can do this type of prep in your sleep. What am I talking about? Plasmid preps, of course!

Plasmid DNA isolation is so routine today in labs that you pretty much expect to get DNA back, even when you make a mistake.  But are you getting back only DNA?  It turns out that plasmid preps are the perfect application to demonstrate a basic difference in two methods for DNA quantification: spectrophotometry (Nanodrop) vs. fluorescent dye (Picogreen).

If you want to isolate microbial DNA from environmental water samples, you need to first separate the microbes from the water.   And since size exclusion filtering is one of the easiest methods to isolate microbes from large volumes of water, this is usually the preferred method.  With this in mind the MO BIO PowerWater DNA and RNA Isolation kits were designed for the isolation of nucleic acids from microbes captured on water filters.   These kits contain 5 ml bead tubes that are large enough so that a standard 47 mm water filter can be rolled up and easily slid into the tube.  Virtually any 0.2 or 0.4 micron size exclusion membrane filter will work, with one caveat.   Bacteria, fungi and protists will be captured on the membrane.    However, virus won’t.  Extracting virus from environmental water samples is a bit trickier.

We recently received the following technical question regarding virus in water.  It’s a common question we get here at MO BIO technical support.

Our team came back from the 4th Annual Argonne Soil Metagenomics Conference held in the Chicago area October 3-5 last week and a hot topic of discussion was filamentous fungi.  Seems that ears were burning all over the cosmos as our technical support lines were also lighting up with questions about recommendations for isolation of DNA and RNA from filamentous fungi. So it looks like this tough critter deserves a blog post all its own.

Fungi are a funny breed of microbe.  What other species can range in size from a single cell to the largest known organism on earth encompassing almost 4 square miles of soil (in the Blue Mountains of Oregon)?  That's right: Fungus. Fungus are everywhere, many beneficial, some tasty, and others deadly.  No matter what you think of them, there is no denying that these are the toughest of microbes.

What makes them so tough?

When it comes to isolating DNA and RNA from all kinds of samples, the fastest and most thorough approach is bead beating.  Whether you have microbes, mouse tissues, plant seeds and leaves, or difficult soils, homogenization using beads will break open both cell walls and membranes and release the desired DNA and RNA so it can be isolated and purified.

Today there are many choices for beads and it can get confusing trying to figure out which is best for what you want to do. To make it easier, here we will summarize the characteristics of all of the bead matrices we use at MO BIO Labs and what types of samples we recommend for them.

In a previous article, we discussed in great detail the homogenization conditions  recommended for RNA extraction. Today's discussion will give you an overview of the entire selection of bead choices for both DNA and RNA.

Tips and tricks for isolation of RNA from cells and tissues remains a very popular subject.  I can't tell you how many discoveries in science depend on identifying that elusive low-copy mRNA with the 5 second half-life!

Joking aside, we've covered quite a few of the basics so far.  For example, I've explained how a strong and complete homogenizationin denaturing lysis buffers performed very quickly is the key to denaturing nucleases and releasing the maximal amount of RNA. And we've talked about the options for homogenization of samples, beginning with liquid nitrogen and ending with all the types of beads available for bead beating using high powered instruments.  All the methods work, so the choice comes down to what is available to you and how many samples you have to do in a day. Bead Beating can easily process many samples at one time with equal and efficient lysis, so consistency between preps is maintained.  Methods that are performed one sample at a time, such as mortar and pestle or rotor-stator homogenizers are better if only a few samples are involved but increase the risk of cross-contamination if cleaning the equipment between the samples is required.

The article this week is about a subject we don't receive many questions about, because, quite simply, the method is so straightforward and consistent that it always works. What am I talking about?  I'm talking about isolation of DNA from formalin-fixed paraffin embedded (FFPE) tissues.

What are FFPE tissues?

FFPE samples are derived from tissues (usually suspected tumor samples) that are fixed with formalin to preserve the cytoskeletal and protein structure and then embedded in a type of paraffin wax so the tissue can be sliced on a microtome, an instrument used to prepare very fine slices, 5-10 microns thick.  Formalin irreversibly cross-links proteins via the amino groups thus preserving the structural integrity of the cells so they can be stained with dyes used to analyze for abnormalities in the tissue that indicate cancer. However, the effect of these cross-linking fixatives on the nucleic acids is detrimental. Isolation of nucleic acids is impaired by both the paraffin wax and the cross-links that block DNA polymerases and inhibit PCR if they are not removed

Since Next Generation Sequencing (NGS) was introduced onto the market less than a decade ago, the technology has undergone rapid growth and improvement. Run speeds have increased, costs have gone down, and the sheer number of bases sequenced per run has improved so significantly that NGS is now within reach for most researchers. Consequently, scores of scientists are trying NGS for the first time. And they have a lot of questions, some of which have been thrown our way here at technical support. The most common question we get is whether or not DNA isolated using a particular MO BIO DNA Isolation kit is suitable for Next Generation Sequencing.

This weeks technical tip is a great question because, as we are all aware, no two environmental samples are ever quite alike. And when working with microbes, they exist anywhere and everywhere, so the substrate often complicates the matter. That's why choosing a method for DNA isolation can be confusing. Luckily, here at MO BIO, we've seen and heard it all and we know what to do.

Take this question, for example.....

Hi MO BIO Technical Support,

I'm considering using one of your PowerSoil kits for an experiment I'm doing. I'm not actually extracting from soil, however. I'm extracting fungal DNA from wood and paper that I've allowed to be colonized by fungi in the environment.

It sounds like combining the Powersoil kit with the Powerlyzer bead-beating would take care of fungal cell disruption and humics from the wood & paper.

However, I'm concerned about polysaccharides and polyphenolics, which may potentially be present in high concentrations in these samples. Would "inhibitor removal technology" be able to remove these inhibitors? Could you fill me in as to whether this kit would be good to use on substrates that might contain high amounts of polysaccharides and polyphenolics? If so, are any changes to the protocol provided for soil necessary?

Thanks for your help.

A. Vortex

B. PowerLyzer

C. FastPrep

D. Precellys

E. All of the Above......

The answer is E! MO BIO Kits work with everything!

The tech tip for today is a great question!  Many people ask us about the adaptability of our kits with all the various bead beaters. Everything we make is compatible with any bead beater on the market. Read more.....

Hello,

I am interested to use the PowerSoil® DNA Isolation Kit. I want to use it with a FastPrep. Is that a problem? Are your tubes adapted for this machine as well?

Best regards,

PhD student

Scientists often come to us with their dirty little DNA problems.   Samples like soil, feces, and blood (oh my!) can make extracting DNA challenging because they are high in compounds like humic acids, polysaccharides, heme, or dyes.  These bind to the DNA and inhibit enzymes used in downstream applications like PCR and sequencing.   MO BIO uses patented Inhibitor Removal Technology® (IRT), a method to remove these substances in many of our kits.   It is very effective at removing the inhibitory compounds without significantly decreasing DNA yield.   But how well does it really work?   Good scientists want to see the data. Recently we received the following request...

Welcome back readers! After a short break from blogging, we're back with some new and different ideas to share with you. We'll start off with a new weekly feature called Tech Tip Thursday (#MBTTT on twitter) where we share some of our customer's questions and our answers with you. What better way of saving you time for life then to post a few of the frequently asked questions we get from all the scientists out there using our products and our answers to those questions? We figured you'd agree.

This week I'd like to share a question about the storage of environmental samples in ethanol as a preservative and why this is NOT a good idea. 

Welcome back readers! After a short break from blogging, we're back with some new and different ideas to share with you. We'll start off with a new weekly feature called Tech Tip Thursday (#MBTTT on twitter) where we share some of our customer's questions and our answers with you. What better way of saving you time for life then to post a few of the frequently asked questions we get from all the scientists out there using our products and our answers to those questions? We figured you'd agree.

This week I'd like to share a question about the storage of environmental samples in ethanol as a preservative and why this is NOT a good idea. Read on....

It's an incredibly handy detachable horizontal tube holder for efficient and consistent mixing of multiple samples for time periods too inconvenient to hold by hand. Try saying that three times fast!

Every once in a while we get technical calls or emails about attaching the vortex adapter to the Vortex Genie 2. It can be a little bit tricky if you never realized that the standard factory attached cup on top is easily removed and can be replaced with alternative adapters for holding different sized tubes. Where can you find these alternative adapters for the Vortex Genie 2? From MO BIO Labs, of course!

MO BIO Labs manufactures adapters in a variety of tube sizes from 2 ml up to 50 ml for the purpose of  hands-free vortexing. If you've never used them before, then this short but catchy instructional video should make it completely clear how to pop the adapters on and off.

Take a look!

They look like the two examples below, which were named Best Student Posters at the 3rd Annual Argonne Soil Metagenomics Workshop held October 5-7 at the Indian Lakes Resort just outside of Chicago, IL.

Organized by Dion Antonopoulos, Folker Meyer, and Jack Gilbert and their staff, postdoc Sarah O'Brien from Dion Antonopoulos' lab, administrator Darlyn Mishur, computational biologists, Elizabeth Glass Ph.D. and postdoc Kevin Keegan from Folker Meyer's lab, and research technician Sarah Owens, from Jack Gilbert's lab,  this was a great three day conference with presentations by leading researchers in the field of environmental microbiology and a keynote lecture by the always impressive Professor James Prosser.

Isolation of RNA, no matter what the source, is nerve wracking, but especially when samples are limited or irreplaceable.  Because RNA is so labile, working quickly but carefully is the key. There are ways to protect your RNA during the procedure so that you can work at a relaxed pace and without so much anxiety. Today I'd like to give you some tips and tricks for isolation of RNA that will help you work smarter, faster and increase your overall success.

1. Chemical Protection

The protection of the RNA begins at the very first step, and this is the homogenization step. You may have noticed that many isolation protocols have you add beta mercaptoethanol (BME) to the lysis buffer. BME is a reducing agent that permanently denatures RNases. So while the smell might keep the sales people out of your lab for the duration of the protocol, you don't want to skip adding this unless you are working with a low complexity sample, such as tissue culture cells. Tissue samples can have a much higher level of nuclease activity so it is best to add the BME.

Many thanks to Russell Neches from the Jonathan Eisen Lab at UC Davis for sending us this great instructional video for using sterivex water filters in the field. And many thanks for giving MO BIO and the PowerWater Sterivex DNA Isolation Kit a shout out in the comments!

Here it is, if you've never used a sterivex water filter before, watch Russell demonstrate how to set it up and collect your samples.

To read more from Russell check out his blog too.

Water filtering gizmo for Sterivex filters

By Mark Brolaski

This summer my wife and I planned to teach our 16 year old daughter the value of having a summer job. Our goal was to teach her to understand that money is fun to have and even more fun if you have earned it yourself and can therefore spend it how you choose (within limits of course, after all she is a teenager!).

The plan was to send her off to the local stores and restaurants to apply and get a job all on her own. This is how we both got our first jobs. Unfortunately, after weeks of applications and turn downs due to lack of experience and an overabundance of other high school kids and recent college graduates applying for the same jobs, the situation looked rather bleak.

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We've been working hard this last month in preparation for the 2011 ASM meeting that just passed. It was a great event and we very much enjoyed meeting everyone at our booth. We love talking to you and hearing feedback and comments about the products.

At ASM this year we introduced a new product to the environmental microbiology community: The PowerWater Sterivex DNA Isolation Kit

Today I'd like to tell you more about this cool product and why we developed it.

What are Sterivex Water Filters?

Sterivex water filters are a great way to collect water samples in the field. These little units are lightweight and enclosed so after filtering water it can be sealed with caps, stored dry, and tossed into a cooler for transport.  Unlike flat filter membranes that need to be transferred to a bead tube for transport and storage, these are self-contained units that do not require any handling......that is until you get it back to the lab.

The weather is finally getting warm which means that we all want to spend more time outdoors getting UV irradiated by natural sunlight instead of under the fluorescent lightbulbs of our labs. That means saving time and working efficiently is of critical importance!

Now is the perfect time to streamline your workflow and find ways to do more with less effort. To do that, I am going to give you an overview of all the cool and convenient lab essentials we carry that will save you time with your routine experiments and get you outside before sunset.

A frequent question to our technical support team is how to isolate DNA from buccal swabs or swabbed material.  Here are our recommendations for performing an extraction of DNA from swabs based on feedback from our customers.  Whether or not to use bead beating depends on whether you are trying to isolate DNA from microbes or human (or host animal) cells.

The eukaryotic cells of the host will lyse easily with guanidine containing lysis buffers and proteinase K. These do not need to be mechanically broken open. However, microbial DNA isolation does need the force of mechanical lysis to obtain the optimal yields. So for this reason, we have several recommendations for working with swabs, depending on your sample type and how dirty it may be.

As we've discussed before, the labs participating in the Human Microbiome Project  (HMP) and now the Earth Microbiome Project (EMP) use the PowerSoil DNA Isolation Kit for extraction of DNA from difficult samples because of the increased performance of the DNA in sensitive enzymatic applications.  Human samples from the gut/stool, surface of the skin, and mouth as well as environmental samples including soils and water are rich in PCR inhibiting substances that are successfully removed using IRT.

Projects like the HMP and EMP involve collecting thousands of samples from hundreds of participants or collection of soil from remote areas of the earth, and hence are irreplaceable. And while these samples remain in storage awaiting processing, changes to the microbial profile may occur. 

For other labs, profiling the microbial content in agricultural lands in response to treatments such as fertilizers, moisture, and nutrients becomes paramount to solving issues with crop production. Vast areas of land must be sampled and analyzed to obtain a broad overview of the microbial diversity in the soil.

PCR is one of the most common techniques performed in virtually all molecular labs today. It is so routine, that when something goes wrong, it can be exceptionally frustrating.  No one wants to spend time troubleshooting a problem that is as simple as mixing a few solutions together in a tube and putting it into a machine.  We need fast answers so we can go on with our research.

Recently in our labs, we encountered unexpected problems while doing qPCR and PCR experiments. As a result, we were reminded of some valuable lessons. I would like to impart them to you here today along with additional advice for troubleshooting PCR problems that usually crop up when you least expect it.

Before I begin with my story, I should explain that we use the Kapa Fast enzymes. We love these enzymes because the PCR and qPCR kits finish in about 45 minutes and the enzymes are so robust that they produce more PCR product compared to other enzymes we've used. So of course, getting a negative PCR or low PCR efficiency is never a problem

This month we talked with Dr. Christine Salomon, Assistant Professor at the Center for Drug Design in the University of Minnesota.  Dr. Salomon’s work intersects chemistry and microbial ecology in the hunt for interesting new natural products with far reaching potential applications.  While much effort has been expended in searching for organisms on the surface of our planet, the deep continental biosphere is relatively unknown. Dr. Salomon is investigating these mysterious microbes half a mile under the surface in the Soudan Underground Mine in northern Minnesota.  A former iron mine, the Soudan Mine is now part of the Minnesota State Park system and an untapped research resource.

Q:  How does the microbial ecology of the Soudan Underground Mine differ from other underground caves?

Today I wanted to talk about a method that you’ve been doing forever in your science career. Something so basic, so easy, that I bet you don’t even have to think about it. I bet you can do this type of prep in your sleep. What am I talking about? Plasmid preps, of course!

Plasmid DNA isolation is so routine today in labs that you pretty much expect to get DNA back, even when you make a mistake.  But are you getting back only DNA?  It turns out that plasmid preps are the perfect application to demonstrate a basic difference in two methods for DNA quantification: spectrophotometry (Nanodrop) vs. fluorescent dye (Picogreen).

Like most labs, we use the Nanodrop to quantify nucleic acids. It is easy, sensitive, and no standard curve is needed. However, when we compared the quantification results from plasmid preps using the Nanodrop and Picogreen, between our plasmid kit and a competitor, the results were very surprising.  But before we go any further....

As part of our research on the best practices for soil microbial DNA extraction, we collect a wide variety of samples for product development. So when we were developing the protocols for the PowerLyzer, we wanted a protocol that worked for most of the samples tested.  Our work on homogenization and bead tubes previously showed that depending on the soil, sometimes a different bead type could give you an increased yield of DNA.  We decided to do a similar study using the PowerLyzer to ask the question: what is the difference in DNA yields and integrity using high powered bead beating between two different soils using the same protocols?  It is not uncommon for people to simply adopt a protocol from a paper for their soil type without doing any optimization.  But, does one protocol really work best for every soil?

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During the ISME conference this past summer I had the opportunity to meet Richard White, and since then, we regularly converse about our research and improved techniques for some of his difficult and unusual samples. Rick is a graduate student in the lab of Curtis Suttle at the University of British Columbia studying microbial diversity in Pavilion Lake Mars Analog site. An area of expertise for Rick is digital PCR. As part of our discussion, he sent me a cool new paper just published from his previous lab, Dr. Stephen Quake and Paul Blainey, on a new technique they developed called digital MDA (dMDA). The paper, "Digital MDA for enumeration of total nucleic acid contamination" was published in Nucleic Acids Research Advance Access November 11, 2010.

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It is a really interesting piece of work that directly addresses some of the questions people have regarding using MDA for single cell amplification of bacterial genomes. I thought it would make a great topic for discussion so below is a review of this paper and the findings. But first, let's go over the basics.....

Metagenomic analysis of environmental diversity is an area of research that continues to rapidly expand in microbiology. Approaches to analyze DNA or RNA samples from soil, water, air, and biofilms involve amplification of the entire 16S rRNA sequences and then analysis of the content and abundance of microbes present.  Many researchers are using next generation sequencing for identification of the microbial diversity in samples. However, the drawbacks to this technique are the expense, the bias towards the most dominant organisms, and the wait time to get your turn at the local core lab sequencer, if you even have access to one of these instruments.

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Another approach to answering these questions hails from the labs of Lawrence Berkeley in the Earth Science.

We've all learned how to isolate RNA from tissues, using methods we've discussed before in this blog and utilizing our fast and easy column purification based kit.

However, we frequently get requests for isolation of microRNA (miRNA) from tissue samples. MicroRNAs are very short 22 base sequences that play a role in regulating gene expression of mRNA by binding to sequences in the 3' untranslated region of transcripts, usually resulting in silencing.  A single miRNA can repress hundreds of mRNAs (1) and mistakes in expression of miRNAs are linked to disease.  Because of their important role in down regulation of gene expression, a current research focus for miRNA is their use in treating cancer (2).

MO BIO recently launched a method for the isolation of DNA and RNA from a very difficult sample type called biofilm. In a previous article, we explained some of the difficulties associated with working with biofilm and biomats. These sample types are not like any other environmental sample and require specialized chemistry to break through the EPS and liberate cells for lysis. 

During the ISME 13 conference in Seattle, WA, last week, the most common question we received from the scientists visiting our booth was: What is the difference between PowerSoil and the new PowerBiofilm DNA Kit?

Today I will explain in greater detail for our readers how these two innovative and specialized methods differ and how they are the same.

When it comes to isolating DNA and RNA from all kinds of samples, the fastest and most thorough approach is high speed bead beating.  Whether you have microbes, mouse tissues, plant seeds and leaves, or difficult soils, homogenization using beads will break open cell walls and membranes and release the desired DNA and RNA so it can be isolated and purified.

Today there are many choices for beads to use and it can get confusing trying to figure out which is best for what you want to do. To make that easier for you, I am going to summarize the characteristics of all of the bead matrices we use at MO BIO Labs and what types of samples we recommend for them.

In a previous article, we discussed in great detail the homogenization conditions  recommended for RNA extraction. Today's discussion will give you an overview of the entire selection of bead choices for both DNA and RNA.

I spend a lot of time reading internet news and blogs to stay on top of current research. In the course of my web surfing, I have found some really great scientific sites for microbiology news and fun. I wanted to share with you my favorite places to visit and let me know what sites you like.

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These are not in any particular order except for news, followed by fun, followed by science gifts and toys.

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Blogs, News, and Information:

1. MicrobeWorld: Microbeworld is a site that compiles the most up-to-date research news from all over the world onto one site. MicrobeWorld is a division of ASM with a dedicated team that scours the internet finding every new microbiology report daily. Also, they've told me that all of you should feel free to submit links to your own blogs or articles, current events in microbiology going on at your institution or local news, and even abstracts for microbiology meetings. I also love their "featured image" on the left.  Submit images from your own research and you can be featured in that spot. (The Myspace page has some cool pictures and videos also.)

We receive many calls from scientists isolating DNA from fecal samples, so I thought I'd share some tips on what we're currently recommending for stool.

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Stool is a rich microbial habitat that has a unique community to each individual.   The variation in stool samples is caused by many factors. Diet can play a major role in composition and texture. Stool taken from a person with a vegetarian diet may have a higher level of polysaccharides and fiber from plant material, and therefore more inhibitors. Also, the use of antibiotics will disrupt the bacteria living in the gut and cause changes in the sample content and consistency. And of course, some foods, especially those eaten raw, for example, sushi, can impact the gut microbiome.

Next generation sequencing is a powerful method increasing in popularity for use in metagenomic and transcriptomic analysis in environmental microbiology. Compared to Sanger sequencing, next generation allows for sequencing of the complete genomic content of a sample without the need to make clone libraries. Using this technique, microbial community analysis can be performed in a matter of days instead of weeks or months.

One problem with next generation sequencing projects is the handling of massive amounts of sequencing data that must be organized, cleaned up, assembled, and analyzed. Sequencing read lengths using the 454/Roche instrument are between 100-400 bp in length and the sequencing of an entire genome can generate millions of pieces of sequence that must be assembled. 

In the past 100 years we’ve learned that each one of us has unique fingerprints, and unique DNA sequences.  Now through the Human Microbiome Project, we’re learning that every one of us has a unique and identifiable bacterial community not only inside of us, but also growing on our skin as well.  Christian Lauber, a postdoc in the Fierer lab at the University of Colorado talked with us about their recent work to elucidate skin microbiomes.

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Q:  Recently your group published a paper in PNAS where you demonstrated it is possible to link individuals to objects that they have touched by comparing the bacterial community on hands and computer keyboards.  What led you to conduct this study?

A:  In earlier work, we looked at the diversity of bacteria living on different people’s hands, how it changed over short periods of time and what happened to bacterial communities after hand washing and treatment with Ethanol.  Surprisingly we discovered that every person we tested had a unique bacterial community that would re-establish itself within 2 hours of hand washing.  Even when hands were washed with Ethanol, those bacteria would regrow relatively quickly without much change.  Given the resilience of these bacteria we surmised that those bacteria would likely transfer to objects that are frequently touched by an individual.

This week we've had a lot of calls and emails from people going down to the Gulf to collect oil contaminated water samples, all asking for the same advice: how do I store water filters collected in the field for later extraction of DNA when I get back to the lab? What is the best practice?  Today's article will share our advice based on the research we have performed in this area. Here is an email from a researcher at the University of Georgia sent to us this week and our answer.

Question:

I am looking for suggestions for storing filters for subsequent DNA extraction using your PowerWater kit. We are heading out this weekend to the Gulf of Mexico and Florida Keys and will be collecting water samples for microbial analysis. We will be able to filter in the field but not complete the extraction process. Additionally our freezer space is limited. Do you have suggestions for storage of the filters that is not dependent on freezing. Is EtOH a possibility (will it work with the extraction process downstream?)  This is a last minute trip and we are trying to work with materials at hand as best we can.

Thanks for your insight.

Most of you are probably back to work after the ASM conference in San Diego. It was a great conference with a lot of exciting talks and posters and we hope you enjoyed our beautiful city.

We would like to send out some special thanks to researchers who collaborated with us to make this one of MO BIO's best meetings ever.

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Seminar presentation:

MO BIO would like to thank Dr. Janet Jansson for an excellent seminar Monday evening at the Hyatt. Her breadth of projects spanning from Crohn's disease to the oil spill in the gulf makes one wonder if she ever sleeps.

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Poster presentations:

We would also like to thank the labs that collaborated with us to generate data for posters we presented.

One of Dr. Janet Jansson's many ongoing projects, and a big focus in microbiology over the last few years has been the study of the human gut microbiome. As ASM 2010 draws near, you can be sure that human microbiome research is an area that will get a lot of attention.

I thought it would be a good time to review a human microbiota paper for all our readers. As an introduction to our guest speaker at our ASM Workshop on Monday, May 24th, this article discusses  Dr. Jansson's 2007 study titled: "Molecular Fingerprinting of the Fecal Microbiota of Children Raised According to Differet Lifestyles", from AEM, vol. 73, No.7, p.2284-2289.

Last month, MO BIO Laboratories was fortunate enough to receive a visit from an exceptionally interesting scientist. Dr. Laurie Connell, of the University of Maine, is involved in a number of research projects spanning from the development of field detection instruments for the detrimental potato wart, to the analysis of paralytic shellfish toxins, to the extreme microbial habitats at the southern most region of our planet. Along with a team of collaborators, Dr. Connell has taken scientific journeys to Antarctica since 1990. She shared images and stories from these Antarctic trips, that at times seem other-worldly. Feel free to throw on a coat as you read on

Many of our readers will attend at least one scientific meeting this year and present a talk or a poster.  This week while reading Microbe World, I came across a link to a great and informative article written by Julian Davies on the ASM Blog Small Things Considered called The Attendee's Guide to Scientific Meetings (part II).

With ASM around the corner, I wanted to share this expert advice on giving a talk or poster at a scientific meeting with The Culture Dish readers.

Below is a short summary of his sage advice.  I highly recommend everyone attending a scientific conference check out the full article for a deeper discussion on how to get the most out of your scientific conference.

In a previous article, we discussed the basic characteristics of biofilm samples and factors that influence sample prep and handling. Today we want to share with you some very important tips for isolation of DNA or RNA from biofilm samples.  After working with numerous different biofilms and biomats, these recommendations are based on our experience and the experiences of the scientists we worked with while developing the PowerBiofilm kit.

This list may expand over time as we continue to learn more about biofilm and work with new and interesting samples. We are always looking for new samples to try in our labs. If you have a sample that is very difficult to work with, let us know and we can try it out and make some custom recommendations.

Here is our current list of Do's and Don'ts for working with biofilm:

Here are the answers to last week's microbiology trivia game. Thanks to all who played. Your t-shirts are on the way. Have a great week!

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Enjoy this scientific spring scavenger hunt and find the answers to these 10 questions on the history of microbiology.  The birth of microbiology revolved around many key discoveries beginning around the mid to late 1600's. These discoveries were at the cutting edge of science at the time and thanks to these dedicated people, the groundwork was laid for discoveries today.

Our last three soil articles were focused on how to get DNA from soil. In great detail we covered the topics of homogenization and the chemistry (and specifically the lysis solutions) needed to isolate high yields and purity DNA.   Working with DNA is far less stressful compared to working with RNA.  Yields are always higher and there is no worry about degradation.

RNA however...

This week's article is an overview about a subject that is near and dear to our hearts here at MO BIO. That subject is BIOFILM!

After spending many months working with all types of biofilms and biomat samples from around San Diego and speaking with researchers all over the world, we understand the difficulty in determining the microbial diversity in these sample types.

In many ways, biofilms are similar to soils in that they are mixed microbial communities, with varying degrees of cell densities, moisture content, chemical composition and inhibitors.  Much like soils they can contain humic substances, metals, and salts, not to mention the polysaccharides, all of which can impact isolation and purification of nucleic acids.  However, the basic structural components of soils and biofilms are vastly different and require different approaches for optimized recovery of DNA and RNA.    

Here we describe some of the things to consider when working with biofilms.

In a previous article, we reviewed a paper that investigated the microbial communities living in showerhead biofilms coming from the municipal water supply. This week, we'll take another look at what's living in municipal drinking water and this time in Phoenix, Arizona.  This new 2010 paper from the Journal of Environmental Quality takes a close look at the presence of a pathogenic amoeba, Naegleria fowleri, in drinking water from various wells and at different times of year . The authors find a new link between the bacterial community of the water in relation to the presence or absence of N. fowleri amoeba.

I thought this would be a good week to help set the record straight on some popular misbeliefs about quantifying DNA (and RNA) yields and purity and especially when working with environmental samples like soil.

Problems achieving high yield and purity are exaggerated in environmental samples because of the added complexity of  microorganism lysis and inhibitor removal.  Quantifying the nucleic acids in these samples is the easy part. But if you don't know what to look for, you can easily make mistakes in interpreting the results, which can lead to a lot of repeat work or missed critical information in your experiments.

Let's discuss some of the common misconceptions surrounding DNA isolation and quanification and what problems to look out for before going to the next step with your sample.

Today we'll pick up where we left off with our discussion on how to maximize the yields of DNA from soil. We have now thoroughly covered the subject of sample homogenization and the importance of the bead type, equipment, and lysis buffer. You can see that the lysis step is the most involved and the best place for optimizing yields.

Next, the sample needs to be cleaned before final purification. This is done using the Inhibitor Removal Technology (IRT) process. Continuing our article at the second stage of the protocol, inhibitor removal, let’s go into greater detail on the different aspects of the chemistry used for the purification of DNA from soil using silica spin filters.

In a previous article I wrote a short primer on soil molecular biology, introducing some of the issues surrounding isolation of DNA and RNA from soil samples, such as PCR inhibitors and lysis considerations. Today I will go into greater detail on the isolation of DNA from microbes in soil and how to optimize your results using the MO BIO PowerSoil^® DNA Isolation Kit.

Because of the amount of detail I want to share with you, this will be a two part blog. The first part will focus on the steps involved in homogenization, that being the beads, the homogenization equipment, and the lysis buffer. In the second part of the article, we will focus on the role of the chemistry in achieving optimal binding, washing, and eluting clean DNA.

How many of you run into problems of false- positive PCR or lack of sensitivity in qPCR when trying to use 16S primers because of the background genomic DNA in your PCR enzyme mixes?

Well, then check out this paper by a team on Switzerland on how to remove residual contaminating genomic DNA from the enzyme mix, reported in the November 2009 issue of Journal of Microbiological Methods. The article is titled: Development and clinical validation of a modified broad-range 16S rDNA PCR for diagnostic purposes in clinical microbiology, and is authored by Abdessalam Cherkaoui, Stephane Emonet, Dimitri Ceroni, Bruno Candolfi, Jonathan Hibbs, Patrice Francois, and Jacques Schrenzel, from the University of Geneva Hospitals in Switzerland.

Oak Ridge, Tennessee is famously known as the base of the Manhattan Project in the 1940’s which ultimately led to the development of the atomic bomb. Uranium enrichment activities on the Oak Ridge Reservation in the 1940s until the 1980s led to substantial contamination in nearby ponds that were used to dump waste. Since then the uranium and nitrate contamination has spread through the ground and now covers an area of about 7 km.

This month we’ve interviewed Dr. Stefan Green, a research faculty member in the Oceanography Department at Florida State University who is working with Dr. Joel Kostka on developing microbial remediation solutions for this site. The overall project, known as the Oak Ridge Integrated Field Research Challenge, is headquartered at the Oak Ridge National Laboratory (Scott Brooks, Principal Investigator) and funded by the US Department of Energy’s Environmental Remediation Sciences Program.

One of the most difficult sample types we work with in our labs is soil. When developing products and methods for isolation of microbial DNA and RNA from soil, we have to take into account the wide diversity of soils in regards to their organic content, texture, pH, and where the soil was collected. These factors and more impact the microbial load and therefore, the yields of DNA and RNA that can be obtained. 

In this article, I’ll dish the dirt on some of the technologies available for isolating DNA and RNA from soil. For more information on the types of organisms found in soil, here is a short primer on soil microbiology.

As Halloween approaches, I wanted to offer some juicy advice that will be of interest to the vampires, as well as the scientists, amongst our readership. I want to talk about blood [cue: thunder, lightning and evil laughter].

Whether you consider it a tasty treat, or a data-rich sample, blood, human or animal, is different from anything else you’ll work with. It is a complicated matrix of cells, plasma, and protein. Human erythrocytes number around 5 x 10⁹ cells per ml of blood, but because they do not have a nucleus, they contain no DNA (avian erythrocytes are nucleated and do indeed contain DNA, hence the need to start with 10 fold less blood for DNA extractions). And hemoglobin levels average around 150 mg/ml of blood. This high level of protein is a major issue in DNA and RNA contamination and PCR inhibition. The combination of cellular debris and protein make this sample as heavenly as candy corn for blood-sucking vampires, but not so for molecular biologists.

We speak with many scientists each week who are working with filtered water for the isolation of microbial DNA and RNA. Water samples are difficult because of their typically low biomass (depending on the water source) and because these samples are precious.

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Why is molecular research on microbes in water difficult?

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For some people, getting back to the original source of water may not be possible for months or even years. For example, we talk to scientists collecting samples at hydrothermal vents in the middle of the ocean, in the Antarctic, and in the Baltic Sea.  For some researchers, water samples may have been collected after a certain event, such as a flood or heavy rain and so the conditions of the water will not be the same in a week or even after a day. They need to get answers from every sample collected and they need it to be accurately reflect the current microbial content.

How many of us dare to look up and actually take notice of the cleanliness of the showerhead during our morning shower? How often do you ever take a little Clorox to the showerhead?

Well according to a study published in PNAS this month (PNAS, Sep 2009; 106: 16393 - 16399) by Leah M. Feazel, Laura K. Baumgartner, Kristen L. Peterson, Daniel N. Frank, J. Kirk Harris, and Norman R. Pace, titled: Opportunistic pathogens enriched in showerhead biofilms, the showerhead is a wonderfully warm, moist, and dark niche perfect for growth of microbial communities.

Why is this a problem?

The dangers of global warming, due in part to increasing carbon dioxide resulting from human activities, are well documented. And the race is on to find technologies to combat it.

Microbialites are one such technology and are the research focus of Dr. Jamie Foster, a dedicated microbiologist at the University of Florida Space Life Sciences Lab at the Kennedy Space Center.

Microbialites play a very important role in scrubbing carbon dioxide from the oceans and converting it to oxygen and calcium carbonate. We had the opportunity to interview Dr. Foster to find out more about her research and the impact it will have on our planet.....