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.

Choosing a Filter:

People who want to determine the microbial communities of collected water will filter them onto filter membranes. The typical size is a 47 mm membrane. This is large enough to have a good flow but small enough to work for DNA or RNA extraction. If the membrane is too small (25 mm), it may clog if the water contains higher levels of debris and if it is too big (142 mm), it will need to be sliced up in order to fit in standard 5 ml and 15 ml tubes.  Ideally, the less handling and manipulations going on with the water filter, the more microbial DNA and RNA can be recovered.

To help make sure that the 47 mm filter membranes are extracted the most efficiently without needing to be sliced into small pieces, MO BIO Labs uses a 5 ml screw cap tube (see picture right). This tube allows for full access of the microbial side of the filter to be homogenized with the garnet grinding resin. We have found after thorough testing that this tube allows for maximal recovery of DNA from all types of filter membranes.

Another question we hear from customers is how to choose a type of membrane. There are many choices from polyethersulfone (PES) to mixed cellulose esther, MCE (cellulose acetate and cellulose nitrate) to polycarbonate to aluminum oxide. Each of these membrane types handle a bit differently and will give slightly different results after extraction.  It is important to remember that the different characteristics of a membrane also reflect its use for other applications such as direct culturing (PES, MCE) or light and electron microscopy (polycarbonate, aluminum oxide).  Overall selection of a membrane for DNA and RNA isolation is more dependent on pore size, sample volume, and retention of inhibitors such as pesticides.  In other words, more than one membrane type may work for your application.

In our experience here is what we found:

Polyethersulfone: Are one of the toughest membranes and can be handled more than the others. They dry quickly under vacuum making them easy to fold without tearing.   Both 0.45 and 0.22 micron pore sizes can be used but a 0.22 micron pore size is best when you want to filter large volumes of water with low microbial biomass because they can handle the longer harder pressure of the vacuum. For nucleic acid extraction, we can get yields equivalent to the mixed cellulose esther with the PowerWater® DNA and RNA Isolation Kits.

Mixed cellulose esther (cellulose acetate and cellulose nitrate): Are best for when a 0.45 micron pore size is needed.  We recommend the use 0.45 micron pore size if your water has a lot of debris and tends to clog or filter very slowly with 0.22 micron pore sized membrane. Cellulose membranes tend to retain water making them a little more difficult to handle.  The video below will demonstrate how we handle them in our lab.

There are several published studies demonstrating that pesticides and herbicides can bind to cellulose acetate and cellulose nitrate so if you are using water that may contain pesticides and herbicides, avoid using cellulose membranes.

Polycarbonate: This type of filter can be more difficult to work with due to its thinness and the ease at which it can wrinkle.  A 0.45 micron pore size is commonly used to prevent clogging.  Unlike the PES and MCE membranes, microbes in your water sample will sit on top of the membrane rather then inside.  This leads to clogging faster but also retention of smaller particles that would have been able to pass through.  We have found that for isolating DNA, less extreme bead beating will give you higher molecular weight DNA. If your sample is used for PCR only, then the stronger bead methods should be fine although expect a lot of shearing.

Aluminum Oxide: This type of filter is also known as an Anodisc™ filter membrane (Whatman).   It handles like a thin sheet of glass and will break up easily in any bead tube. Most labs are not using these due to the difficulty in transferring them to storage tubes. These are used with samples containing very low biomass such as ocean water.  They come in both 0.45 and 0.22 micron sizes.  Similar to the polycarbonate, microbes are retained more on top rather than within the filter, leading to easy extraction of DNA and RNA but also increased shearing with bead beating.

How to Handle a Filter Membrane:

Many of you out there probably already have a good technique for folding your filter membranes and placing them into a tube. For those of you who are new, or experiencing problems with this, here is a video we made in our lab to demonstrate the technique. This is a 0.45 micron mixed cellulose esther membrane.  Demonstrating is Heather Callahan, Ph.D, the scientist who created the PowerWater^® kits.

Summary:

We know how hard it is to work with different environmental water samples and how important it is to get every last drop of DNA or RNA. Our goal is to make sure you are successful at every step. When we developed the PowerWater and RapidWater products, we kept that in mind as we optimized each step; from the tube needed to grind in, to the matrix used for grinding, to the solutions used for removing inhibitors, to the binding chemistry, to the washing chemistry, and the final elution. If you have any questions on how to maximize your water filter DNA or RNA extraction, please send them to us and we’ll get back to you.  I can assure you, if you have a problem, we have the answer.

Ask the Water DNA and RNA Isolation Experts!

For more information on Water DNA and RNA Isolation products, go to:

PowerWater^® DNA Isolation Kits and RapidWater^® DNA Isolation Kits

PowerWater® RNA Isolation Kits

*This article is a re-post of the original which was published in October of 2009

Fun fact: Shakespeare's sonnets have been encoded in DNA

I would like to meet whoever is responsible for perpetuating the myth that scientists are a stuffy and dry bunch and introduce him or her to the amazingly creative and artistic scientists we have the pleasure of interacting with at MO BIO.  This month we invited our users to submit a poem about their MO BIO experience. The results were quite impressive. The topics ranged from our kit’s unparalleled ability to remove inhibitors to our great customer service. In return for their very kind words, we offered up a t-shirt and discount code.

Be sure to check out the video links of MO BIO employee’s reading the submitted poems.

**************** Read the rest of this entry »

Who hasn’t pondered the age old question: Which came first, the chicken or the egg?

Debates on this subject have kept philosophers busy for centuries, lending to rich discussions on everything from evolution of chickens to the beginning of life and the universe. One could ponder this question from a literal point of view and discuss the evolution of egg laying species and whether or not this pre-dates the appearance of chickens. Or one could set forth on a metaphysical journey and focus on the possibilities of how one life form can exist without its developmental precursor or how a precursor exist without its original maker?

Here at MO BIO, this discussion led us down a different path. The question we propose to you is: Which came first, the DNA or the protein? You need DNA to know what proteins to make and how to make them, but, you need proteins to synthesize more DNA, to transcribe the DNA into RNA, and to hold it all together.

Read the rest of this entry »

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? Read the rest of this entry »

We’ve all got our groove on. Sampling and processing seems like a breeze despite it’s challenges.  The flow of efficiency has finally set in and it feels good! Read the rest of this entry »

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. Read the rest of this entry »

It’s Valentine’s Day and love is in the air at MO BIO! To celebrate this special (not so special for some) day, we thought we’d tell you about the cutest and nerdiest proposal of all time. Watch out guys, if your girlfriend sees this and is expecting a ring sometime soon, the pressure’s on!

You may have heard some buzz floating around recently about the guy who cleverly proposed to his girlfriend via agarose gel. Yes, we know, it’s incredible.  We wanted to ask him a few more questions about this amazing, geeky proposal,  so we tracked down the proposer, Eric (currently a Post-Doc at UCSD) , to get the whole story.

How did you two meet?

We were both graduate students at Stanford. We met at a grad student event and played volleyball together. We ran into each other again later at a department happy hour.

Where did you get the idea from? Read the rest of this entry »

Regional Climate Warming: West of the Antarctic Peninsula
Jan 17, 2013
68°00.2S X 69°30.8W
Sunrise  11:37pm
Sunset  9:49pm

MO BIO Labs is going to collaborate with our friends at the popular science blog site, Bitesize Bio to sponsor a couple of webinars for their readers.  Since MO BIO Labs has a primary focus in microbiology and ecology, we want to sponsor seminars that are going to highlight the research and scientists working in this exciting field.

As I was pondering potential speakers, I thougth about the women in science I’ve heard speak, who are an inspiration to me. But my experience is also limited being in the private sector. I don’t get to interact with as many women thought leaders as I would like. The question then became: who are the leading women in the field right now?  What women scientists do we have to draw insight and inspiration from? Read the rest of this entry »

Boondoggle!
by Emelia DeForce
Jan 10, 2013
66°15.0S X 67°21.9W
West of the Antarctic Peninsula
Sunrise  2:50am
Sunset  12:19pm

I have to pause for a minute to pay homage to and tell you about the humbling power of Palmer Station.  There are only about 30 people that live there in the summer months (Sept-April) but all of them are responsible for running not only a station, but a home.  There are cooks, seamstresses, metal workers, electricians, doctors, scientists, etc. many of them acting in multiple roles on a daily basis.  Each individual plays an integral part to make the station a success even if it means scrubbing someone else’s bathroom floor after lunch.

During turnaround, I was invited on a “boondoggle”, or mini outdoor excursion.  Before any boating activities, one Read the rest of this entry »