Interested in Soil Ecology and Biogeochemistry?

Gain an integrated perspective with world-renowned faculty to address critical questions using current analytical techniques, experimental approaches, and instructional models.  Topics include:

• What are the physical, chemical and biological components of soil?
• What do molecular techniques tell us about soil biodiversity?
• How does soil chemistry affect carbon and nutrient cycling?
• How are soil processes affected by global change?

The Summer Soil Institute is designed for graduate students,post-docs, professionals, faculty, and K-12 teachers. Located at the confluence of the Rocky Mountains and the Great Plains, participants will be provided with hands-on experience with lab, field, and modeling techniques including:

Soil physics and biochemistry
• pedology
• organic matter fractionation
• stable isotopes
• soil respiration
• trace gas fluxes
• NMR, FT-ICR-MS, and XRD

Soil biology-microbes and fauna
• DNA extraction
• quantitative PCR
• enzyme activities
• microscopy-based identification
• soil food web modeling

2012 Faculty
Thomas Borch
Francesca Cotrufo
Gene Kelly
John Moore
Mary Stromberger
Joe von Fischer
Diana Wall
Matthew Wallenstein
Featuring honored guest Dr. Eldor Paul, author of Soil Microbiology, Ecology, and Biochemistry

Where and When
Colorado State University
Fort Collins, CO
July 8-21, 2012

http://soilinstitute.nrel.colostate.edu/

For more information and to apply, please visit the website:
Web: http://soilinstitute.nrel.colostate.edu/

Email: soil@nrel.colostate.edu

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!

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.

Part of the conference activities was a lively poster session covering a wide range of exciting projects at various stages of completion. As a sponsor for the conference, MO BIO had the great pleasure of providing the award for best student poster ($500 in product credit) and a 2nd place award for the runner up ($250 in product credit).   The winning presenters needed to be able to explain their work verbally and answer questions by the judges, as well as have clear concise wording that covered all aspects of scientific communication (introduction, hypothesis, methods, results, and conclusions) on the poster.

Today I would like to showcase the amazing students and their award-winning posters. Whether or not you are in this area of research, you’ll want to take a look at these posters. They exemplify a great way to present your data that does not overwhelm the reader and yet makes it very easy to follow the scientific story.

Poster Title: Soil Microbial Diversity in a Mongolian Climate Change Experiment

Click to view full poster

The winning poster was given by PhD student Aurora MacRae-Crerar (above), who is working in the labs of  Peter Petraitis and Brenda Casper at the University of Pennsylvania in Philadelphia.  It is evident when a student is becoming independent when they can so comfortably discuss their project goals and next steps with the level of enthusiasm demonstrated by Aurora. Very nice job!

Poster Title: Functional Screening of Soil Metagenomic Libraries for Bacterial Conjugation Genes

Click to view full poster

The runner up for the best poster award was undergraduate student Cveta Manassieva (above) from the lab of  Dr. Trevor Charles at the University of Waterloo.  Download her poster and take a look at how well she outlined her scientific method, making it clear and easy to follow with good visuals.  With this format, it is easy to understand what

she is doing and why, with very little effort! Very nice job Cveta!

See You Next Year!

One of the most enjoyable parts of my job is meeting scientists at conferences, talking about your projects, and helping the students get through their PhD work as painlessly as possible.  The Argonne conference was a great chance to have 1-on-1 time with many new and experienced metagenomics researchers, both through the poster sessions and at meal time.  I look forward to meeting many more of you next year as we try to cover as many events as possible in the area of environmental microbiology.

And if you have an event you would like MO BIO to sponsor or offer support in the form of student awards, just drop us an email or comment on the blog and we’ll be in touch!

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.

If your extraction is taking place in the presence of phenol, then the BME can be skipped because the phenol will do the job of nuclease inactivation.

2. Fast Homogenization!

The very next thing you want to do is add the BME containing lysis buffer (or phenol based lysis reagent) to your sample for homogenization.  We’ve discussed lysis and homogenization before, including a thorough overview of bead tubes and sample types.  Bead beating is a great way to homogenize a lot of samples quickly and under the exact same conditions, eliminating a source of variability between preps.

The important point to remember to protect your RNA is that you want to move through this step fast. The tissue sample should go right from the freezer to the bead tube or vessel as quickly as possible. Once the sample is removed from the freezer and begins to thaw, the countdown has begun for RNase activation. 

Before I take my tissues out of the freezer, I set up my bead tubes with lysis buffer and put them in a Stratacooler or a similar chill block and place it by the scale, keeping my tubes cold. The lysis buffer does have a high concentration of guanidine thiocyanate salt which does precipitate when chilled. However, once the sample goes into the bead beater, the heat generated dissolves the salts quickly and the RNA integrity is higher when the bead tubes start out cold.

For my tissue samples, my approach is to freeze aliquots of tissue in RNALater in ~50 mg pieces. I typically use 25 mg per prep so when I thaw one tube of tissue, I know I have two preps worth of sample and I can quickly slice the tissue in half and drop them into pre-chilled bead tubes.  My sample goes from the freezer to the scale and then quickly into a -20C chilled bead tube. Once I have all my samples in bead tubes, I place them in the PowerLyzer where they are homogenized for two cycles at 45 seconds. Any frozen salts quickly dissolve.

The reason fast homogenization is so important is because you want to expose every cell to the guanidine lysis buffer and the BME.  If you have any cell clumps in the lysate, no matter how small, those pieces will have active RNases that can cause low RIN values and degraded looking RNA in your final gel.  Complete homogenization is important.

A hand held polytron or rotor-stator homogenizer may be used for RNA extractions also, and this is especially handy for large scale preps. I can usually get the tissue broken down in about 30 seconds. But all of the other samples are sitting and waiting as one prep at a time is homogenized. Keeping the lysis buffer cold can help but it can’t be frozen before homogenization.

3. Removal of DNA

The rest of the protocol is easy, and as long as the sample was high quality when you started, the RNA will be high quality when it is eluted. So what else is there that could cause degradation or loss of the RNA? That’s right, the DNase step.

DNase digestion is frequently performed on the spin column and this is a great way to save some time on the post extraction processing. However, some samples have so much DNA (for example, spleen and thymus, even some soils) that the on-column DNase is just not efficient for complete removal. In this case, DNase digestion is solution is necessary.

Room Temperature Stable DNase and Removal Resin

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.  And most DNase enzymes are stored frozen and need to be aliquoted to avoid freeze/thaw cycles that can reduce enzyme efficiency.

We came up with a better system that protects the RNA all the way to the final step. RTS DNase is a liquid room temperature stable DNase with very high activity (1 ul of enzyme can digest 30 ug of DNA in 20 minutes). The best part is the clean up step. The DNase comes with a removal resin that binds the enzyme and cations and pulls them out of the RNA sample making it ready to use in qRT-PCR without any inhibitory additives or heat steps.  The resin is so efficient that even with 10 units of enzyme in the reaction, it is completely removed (figure below, lanes 3-4) compared to an alternative resin method where only 2 units of DNase enzyme is used (lanes 1-2).

This means that you can protect your precious RNA as well as hours of work and get better accuracy in gene expression assays.

RTS DNase Removal Resin completely removes DNase. Samples were subjected to DNase treatment and enzyme removal using the RTS DNase™ Kit or a competitor’s kit, and then analyzed for residual DNase activity using the MO BIO DNase-free certification assay. Lane 5 is the negative control and did not receive DNase. Samples were incubated for 1 hour at 37oC, followed by inactivation for 5 minutes at 65oC. Results are shown on a 1% agarose gel. The RTS DNase Removal Resin successfully removed the DNase (lanes 3-4), while the competitor’s resin failed to remove all of the DNase from the samples (lanes 1-2).

Before I summarize, let me tell you about several great promotions you can take advantage of this month if you purchase the RTS DNase Kit Cat# 15200-50.

1. Buy one 15200-50 and get a free timer (while supplies last, see right)
2. Buy two 15200-50 and get a $20 REI gift card (October only)
3. Buy the RNA PowerSoil and RTS DNase™ Bundle and get an additional 10% off. Use cat# 15200-BUNDLE when you place your order.

Samples are available but you have to order in October to take advantage of these special limited time promotions, so don’t wait to make the switch to the RTS DNase and start protecting your RNA right away!

Summary

Working with RNA is easy when you know how to protect against the sources of trouble.  Fast homogenization in a protective lysis buffer is critical and then gentle DNase treatment of the RNA at the end is the icing on the cake.

Yes, certified RNase-free gloves are a great extra to have as well as  UltraClean Lab Cleaner for removal of nucleases from the bench and equipment.   We use these routinely in our labs. But the essential requirement for high quality RNA preps involve the very chemicals and plastics or beads that are going to come in contact with your sample and the RNA. The use of BME, consistent and fast homogenization, and RNase-free DNase with removal resin will be your ticket to success in every prep no matter what the sample.

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

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.

We decided to go with plan B, which was to give her a job at MO BIO. Now you might be thinking, yeah great idea but how does this apply to the average kid without parents in the biotech industry? I would just counter that with, while I agree to some extent, in today’s economy, there really is not a lot of opportunity out there for teenagers and if you can’t help them find a job with your connections, the fact is, they may go the whole summer without any work experience. All well and good from their perspective, but another lost summer where your son or daughter will not know the real value of money, nor will they learn the humility of having to follow orders or risk not keeping their job.

So with Plan B our only option, we decided to hire our daughter as a lab assistant. A bottom of the totem pole position where she would wash lab dishes, prepare agar plates and growth media and if she proved herself, move on to more complex tasks in the lab, like DNA and RNA isolations, gel electrophoresis, and other molecular biology techniques.

We started with a bit of uneasiness that our employees would think “oh yeah, the bosses daughter, better treat her delicately.” So we did a preemptive meeting to make sure everyone treated her just like any other MO BIO employee. No favoritism was expected nor would it be acceptable. To our surprise, after just a few weeks on the job, we started hearing things like “Wow, noob lab tech (name changed to protect the innocent) is really doing a great job. She follows direction and is really catching on very fast.”

To our delight, not only had she made it to work on time every day, followed her supervisors instruction, and shown herself to be a productive member of the MO BIO team, she had also received her first paycheck and deposited it in her first bank account that was not strictly savings. Now, in these times, we need ways to give our kids a sense of self-worth that comes from something other than scoring a game winning goal, a home run, or winning a surf contest (Sorry I had to put that in. As you may or may not know we are from California). A real pay check has a way of boosting self-confidence like nothing else.

Talk about proud parents, and even more important a proud young person seemed to have emerged from the somewhat fragile shell of a 16 year old adolescent. She seemed to walk just a bit taller and smile just a bit more confidently than she had just a few weeks prior.

Another benefit was that she was able to see other women who had made careers in science, and not only were they successful, they were having fun at their jobs. This is such an important time in a teenager’s life because they are beginning to make decisions about career paths and college choices. So to give her the opportunity to see what it means to be in a research and development lab at MO BIO and to see how a person can be creative and produce new products was an invaluable lesson.

I guess the moral of this story is that if there is any way you can possibly get your high school student, friend’s high school aged child, or any other inquisitive young adult you know a summer job in science, please do it. You have so much to gain and the world may benefit from having another young person get interested in this fast growing field we call science.

The more creative, the better the prize. Prizes will be along the lines of free kits, swag, and anything else we can think up (which probably involves chocolate, coffee, or both).

So get out the video camera, have some fun and make us laugh!

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 problem with Sterivex water filters is that they are self-contained and getting the filter out is an arduous task. Some people will break open the plastic casing, typically using a cigar cutter or a hammer, and then remove the filter from the unit for extraction using various methods, such as the PowerWater DNA Kit.

The more common method is to fill the unit with a lysis buffer and whopping amounts of enzyme and digest the microbes in the unit. The solution is then removed with a syringe and processed with phenol-chloroform and ethanol precipitation. The whole process takes anywhere from 24 hrs to 3 days depending on how long you digest the sample.

Research and Development of PowerWater Sterivex

Well, we figured there had to be a better way to do this. So leave it to R&D scientist Heather Callahan (picture left), inventor of the PowerWater, PowerBiofilm, and PowerFood Kits, to figure this out.  It wasn’t a straightforward approach. Getting the microbes out of the filter membrane was tricky.  Unlike flat filter membranes, microbial lysis was difficult in the narrow space of the cartridge.

The turning point was the development of a solution that functions to loosen and release microbial cells caught inside the membrane.  The Cell Release Solution was a major breakthrough in the development because it reduced the amount of time needed for incubating the filters in lysis buffer and reduced the need for excessive bead beating that can be detrimental for DNA integrity in some water samples.  Once we had a way to get cells out of the membrane, we were half way there. There were, however, more challenges.

Because the units require a higher volume of lysis buffer to cover the membrane, this made concentrating the DNA in a mini spin filter a problem.  We needed to take a large volume and concentrate it onto a small capture membrane so the DNA is eluted ready to use and at a workable concentration. To accomplish this, we had to re-invent the loading process.  A new mini spin filter that could handle the flow from a large volume under vacuum was created.  Using a syringe barrel, we could easily load 4 ml of solutions into the column within a few minutes.

These two innovations were the key to taking a 2 day process and turning it into a 40 minute method.

But the seal of approval had to come from our scientist testers.  With the help of several labs that had Sterivex samples in storage or collected fresh samples just for this evaluation, we were able to get the feedback we needed to launch this kit.  Our testers gave it a thumbs up. And with that we introduce to you the newest addition to the MO BIO product family, the PowerWater Sterivex DNA Isolation Kit.

It still has IRT for removing inhibitors and it has a new bead tube for homogenization of microbes if you have some tough critters that need extra mechanical lysis. We developed the main protocol to work for the most difficult to lyse bacteria and then give suggestions for reducing the lysis conditions if your sample doesn’t require it. To show you an example of how well it works, Heather presented her studies comparing the extraction of Bacillus spores to vegetative cells using Sterivex water filters in a poster at ASM.  Here we show the power of the Cell Release Solution in extracting spores in the Sterivex membrane to achieve high yields of DNA in comparison to removing the membrane and extracting directly. It really does work.

Here is the poster for review.

If you have any questions about the method or want to discuss your project, please contact us at technical@mobio.com or leave a comment on this blog and we’ll answer you right here.

We are also very pleased to announce that the undergraduate student who is the primary author of the second poster, Joe Lahti, from the University of Minnesota was awarded an ASM Student Travel Grant for his work. Congrats Joe!!

Below are our abstracts. Let us know if you are presenting a poster or were selected for a talk and used MO BIO products. We would love to come visit you during your session and tell other scientists to check out your work!

Poster #1:

Efficient DNA Extraction and Purification of Vegetative Cells and Spores from Sterivex Filter Units 

H. A. Callahan, S. J. Kennedy, M. N. Brolaski
MO BIO Laboratories., Inc., Carlsbad, CA. 

Background: Sterivex™ filter units (Millipore) are commonly used for collecting microbes from water samples for subsequent nucleic acid isolation. Protocols developed for the extraction of DNA from these units requires either breaking the plastic casing to remove the filter inside for processing or the incubation of lysis solutions inside the unit itself.  Breaking open the plastic casing is both time consuming and requires aseptic handling of the unit and membrane.  Disadvantages of both protocols include the length of time to complete the extraction and the need for phenol-chloroform to clean the lysates followed by alcohol precipitation for purification and concentration of the DNA.  The goal of this project was to develop an in-unit method for Sterivex™ filter unit DNA isolation that was fast and efficient without the use of organic solvents, and then to compare the new method with a previously published protocol.  Methods: A novel membrane pretreatment solution that resulted in release of the bacterial cells from the membrane with high efficiency was developed.  This solution allowed for recovery of bacterial cells so that lysis with bead beating produced yields equivalent to enzymatic lysis.  The results of the new microbial releasing solution combined with chemical lysis and bead beating was compared to an in-unit enzymatic DNA extraction and purification protocol for Bacillus subtilis vegetative cells and spores.  Results: DNA was obtained with both protocols, but the highest DNA yield achieved from both vegetative cells and spores was with the new method.  When the time required for performing both methods side by side was compared, the novel extraction method using the releasing solution required only 40 minutes from start to finish while the enzymatic method required 48 hours. Conclusions: Incorporation of a novel membrane pretreatment solution was equivalent to the use of enzymes for isolation of DNA from microbes in a Sterivex™ filter unit.  The new protocol was performed both rapidly and easily without loss of critical microbial DNA and without the use of organic solvents.  

Poster #2:

Evaluation of Various Filter Membranes for Optimal Quantification of Bacteria
Authors: J. D. LAHTI1, S. J. KENNEDY2, H. A. CALLAHAN2, C. E. SALOMON1
1Univ. of Minnesota, Ctr. for Drug Design, Minneapolis, MN, 2MO BIO Lab., Carlsbad, CA 

Background: Isolation of DNA from microbes collected on filter membranes is a common practice for studies of microbial load and biodiversity in water. Analysis of the microbial content of water requires the collection of large volumes that are concentrated onto a solid membrane support prior to cell lysis and DNA isolation. The membranes commonly used for this purpose can vary based on pore size and membrane composition. The goal of this project was to determine whether differences exist between different types of filter membranes for capture of bacterial cells and the subsequent isolation of DNA. Additionally, the isolation of DNA from cocci and rod shaped cells was compared to explore a potential bias due to cell shape.  Methods:  To evaluate the effects of cell shape on bacterial capture using filter membranes, cultures of Micrococcus luteus and Bacillus subtilis were used as cocci and rod shaped bacteria, respectively. The filter membranes analyzed were composed of cellulose acetate, polyethersulfone (Supor®), polycarbonate, and nylon with a pore size of 0.45 microns. To quantify recovery of bacteria in pure cultures and from membranes, a qPCR assay was employed to detect a single copy gene in each bacterium. Results: M. luteus was detectable down to 10 bacteria/ml in a 100 mL volume from all membranes tested. A reduction in recovery of B. subtilis DNA was observed on all the filters except for polycarbonate. The cellulose acetate membranes had the lowest recovery of B. subtilis DNA with recoveries below 10%. Conclusions: 1- Lysis of gram positive microorganisms from filter membranes requires optimized lysis conditions including heating of filters in lysis buffers prior to bead beating. 2- The detection sensitivity from water filters requires a minimum quantity of 1000 microbes, thus more water should be used for optimal detection of bacteria in low biomass samples (<10 cells/mL). 3- No difference in recovery of DNA was observed for M. luteus bacteria between the different filter types. However, for B. subtilis, the cellulose acetate and nylon membranes demonstrated the lowest percent recovery of bacteria even at the highest cell numbers (10,000 cells). Collectively, these results suggest that filter composition can be optimized for recovery of populations of bacterial species depending on their density and cell shape.

I have been especially impressed with Bradley’s project management skills and his mentorship of the undergraduate student who has been working on the projects. It is clear that Bradley is getting excellent training for a future in academic or industrial science!

On to Bradley’s graduate research. He is currently very focused on ammonia oxidizing archaea in the ocean. His work involves a team of researchers who have collected samples the world over, ranging from Antarctic regions to Canadian fiords to examine newly found archaea. Their goals are to better understand their role in the complex oceanic environment.    Here’s more from Bradley on why this work is so important.

Q:  What are ammonia oxidizing archaea?

A:  In recent years, archaea have been found to be a dominant form of life in the ocean making up nearly 1/3 of all prokaryotic cells in the ocean.  Archaea were once thought to live primarily in extreme environments, but in 1992 they were discovered in marine systems and recent studies have confirmed their abundance.  Further, it appears that some of these marine archaea are actually autotrophs capable of converting ammonia to nitrite as part of the oceanic nitrogen cycle. 

Q:  What is the significance of the nitrogen cycle in the ocean?

A:   Nitrogen is an essential nutrient in both the ocean and on land.  Nitrogen is typically found as nitrogen gas (N2) and ammonia.  The archaea we study then can take this ammonia and convert it into nitrite, which can then be oxidized to nitrate, in order to provide useable forms of nitrogen for higher marine organisms.  Before the discovery of these archaea it was presumed that bacteria were primarily responsible for ammonia oxidation.  Our work investigates the importance of archaea in this process as well as how their role may vary in different oceanic environments.

Q:  Where do you collect samples and how do you analyze them?

A:  We have collected samples from a wide variety of locations including the Southern Ocean off the western Antarctic Peninsula, Monterey Bay, Gulf of Mexico (prior to the oil spill), off the coast of Georgia, and fiords in Canada.  Specifically we are using sequencing and PCR analysis to look at changes in the genes for amoA and 16S rRNA.  amoA is a gene which encodes the enzyme responsible for catalyzing the rate-limiting step in ammonia oxidation.  Because we do not have a pure culture to work with in our lab, we rely primarily on these molecular techniques and also combine them with microscopy and fluorescent in situ hybridization (FISH).

Q:  How have MO BIO’s products helped you carry out your work?

A:  We generally have used phenol-chloroform techniques to isolate DNA from these organisms.  However, we recently began using MO BIO’s PowerWater® DNA Isolation Kit after comparing it to our previous methods and found a greater success rate for extraction of DNA.  Additionally we are collaborating with MO BIO to optimize protocols using the PowerLyzer™ PowerSoil® DNA Isolation kit for related marine sediment and forest soil samples. 

For more information about Bradley’s work, please visit Dr. Hollibaugh’s website at the University of Georgia, Department of Marine Sciences.