Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity

Snowflakes---MA-400x600Over the last few months we have published several blogs about qPCR—from basic pointers on avoiding contamination in these sensitive reactions to a collection of tips for successful qPCR. Today we look in depth at a paper that describes the design and and optimization of a qPCR assay, and in keeping with the season of winter in the Northern hemisphere, it is only fitting that the assay tests for the abundance and identity of ice-nucleating bacteria.

Ice-nucleating bacteria are gram-negative bacteria that occur in the environment and are able to “catalyze” the formation ice crystals at warmer temperatures because of the expression of specific, ice-nucleating proteins on their outer membrane. Ice-nucleating bacteria are found in abundance on crop plants, especially grains, and are estimated to cause one-billion dollars in crop damage from frost in the United States alone.

In addition to their abundance on crop plants, ice-nucleating bacteria are also found on natural vegetation and have been isolated from soil, snow, hail, cloud water, in the air above crops under dry conditions and during rain fall. They have even been isolated from soil, seedlings and snow in remote locations in Antarctica. For the bacteria, ice nucleation may be a method to promote dissemination through rain and snow.

Although ice-nucleating bacteria have been isolated from clouds, ice and rain, little is known about their true contribution to precipitation or other events such as glaciation. Are such bacteria the only source of warm-temperature (above temperatures at which ice crystals form without a catalyst) ice nucleation? Can they trigger precipitation directly? What are the factors that trigger their release from vegetation into the atmosphere? Can we determine their abundance and variety in the environment? Continue reading “Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity”

Extraction of Plant DNA Made Easy

By Trillium1946 at en.wikipedia (Transferred from en.wikipedia) [Public domain], from Wikimedia Commons
My one attempt at working with plant DNA when I was at the lab bench was trying to create a shotgun library from a rice BAC. Never have I needed to isolate nucleic acid from the source material, but based on my conversations with plant scientists, it can be problematic endeavor between the tough tissue and the compounds that can copurify during extraction and inhibit downstream applications. And if you want to isolate DNA or DNA from plant samples in an automated format, that just adds to the difficulties. Here I review an Applications in Plant Sciences article that compares DNA isolation using the Maxwell® 16 System with two other methods on 25 different plant species samples. The authors note that Promega provided the Maxwell® 16 instrument, DNA isolation cartridges and advice on its use. Continue reading “Extraction of Plant DNA Made Easy”

Freedom to Focus: Maxwell® Rapid Sample Concentrator

Wish I had one of these when I was at the lab bench...
Wish I had one of these when I was at the lab bench…

Back in the dark ages, when I was a graduate student, my idea of “automated” plasmid DNA extraction involved performing home-brew, “toothpick preps” in “strip tubes” or , if I was really feeling ambitious, a 96-well plate. I would get just enough DNA to check for the presence of an insert, but the quality of the DNA was too low and the quantity too small to even consider using it for any other downstream experiments like amplification. And increased throughput for other nucleic acid extraction needs? Nope. If I wanted genomic DNA, RNA or high-quality plasmid DNA, I spent time with columns and tubes, giving each sample my undivided individual attention. Remember cesium chloride preps for RNA isolation? Even with the advent of column purification, which greatly simplified and standardized my protocols, nucleic acid purification was still a manual task that required a lot of time and effort to get the high-quality product I needed. Doing the experiments that would answer the questions that I really wanted to ask (those “downstream experiments”), meant spending time at the bench performing careful (if tedious) work to isolate and clean up the highest quality nucleic acid possible. Even then inconsistency in sample prep could wreak havoc on downstream work. Fortunately, for the modern scientist, personal, bench top automation, has progressed far beyond the toothpick and the strip tube to quality-tested, reliable nucleic acid extraction platforms like the Maxwell® Rapid Sample Concentrator (RSC). The Maxwell® RSC improves sample preparation consistency, eliminating variability associated with manual handling, and your downstream results will reflect this consistency.  With the RSC you can extract DNA or RNA from up to 16 samples in approximately 1 hour and viral total nucleic acids in less than an hour. The instrument is easy to use: simply load the sample, push a button and walk away. Cross contamination is minimized and the instrument is supported by the Promega technical support and service you have come to trust over the past 35 years. Do you want to know more about how the Maxwell® RSC can become your research partner, giving you the freedom to focus on the work that interests you the most? Register for the free webinar and see the data for yourself: high-quality nucleic acid that performs well in downstream analyses. You’ll even be able to view videos illustrating RSC  setup and use. Register today for the free webinar.

Who Stole the Cookies? Technical Services Scientists Offer DNA Labs to Area Schools

teaching-lab-2Guest Post from Promega Technical Services Scientist, Caroline Davis.

On a snowy day in January, someone stole the cookies that were to be served with lunch from the Rome Corners Intermediate School cafeteria.   The kids were distraught. What should they do?  Luckily, the Green 2 Team science class was there with Promega’s Technical Service Outreach team (and Paraj Mandrekar, Senior Research Scientist and Green 2 Team Dad) to help.

The students realized that the thief had taken a bite out of a strawberry and left part of it behind, along with his DNA.  After a short discussion on what DNA is and why you would want to isolate DNA, the 6th graders extracted DNA from strawberries using household reagents under the guidance of the Promega scientists.  The students used pipettors, beakers, microfuge tubes and flipper racks, giving the students a glimpse of the tools that scientists use everyday in in a molecular biology lab. Continue reading “Who Stole the Cookies? Technical Services Scientists Offer DNA Labs to Area Schools”

ProK: An Old ‘Pro’ That is Still In The Game

Proteinase K Ribbon Structure ImageSource=RCSB PDB; StructureID=4b5l; DOI=http://dx.doi.org/10.2210/pdb4b5l/pdb;
Proteinase K Ribbon Structure ImageSource=RCSB PDB; StructureID=4b5l; DOI=http://dx.doi.org/10.2210/pdb4b5l/pdb;
If you enter any molecular lab asking to borrow some Proteinase K, lab members are likely to answer: “I know we have it. Let me see where it is”. Sometimes the enzyme will be found to have expired. The lab may also have struggled with power outages or freezer malfunctions in the past. But the lab still decides to keep the enzyme. One may rightly ask – why do labs hang on to Proteinase K even when it has been stored under sub-standard conditions? Continue reading “ProK: An Old ‘Pro’ That is Still In The Game”