The European Union (EU) has a zero tolerance policy for products containing any material from non-authorized genetically modified (GM) crops. Seed entering EU markets may not contain even trace amounts of non-authorized genetically modified material. In 2012, as the global use of GM crops increased, seed testing loads in the EU continued to build. Isolating genomic DNA (gDNA) using traditional manual methods was becoming impractical in the face of increasing amounts of material that required testing. There was a growing need for an automated method to isolate gDNA from seed samples. Working to address this need, a group of scientists from the Bavarian Health and Food Safety Authority collaborated with scientists from Promega Corporation to evaluate the Maxwell® 16 Instrument and the associated chemistry as possible a solution for the testing labs. Continue reading
Bottom-up proteomics focuses on the analysis of protein mixtures after enzymatic digestion of the proteins into peptides. The resulting complex mixture of peptides is analyzed by reverse-phase liquid chromatography (RP-LC) coupled to tandem mass spectrometry (MS/MS). Identification of peptides and subsequently proteins is completed by matching peptide fragment ion spectra to theoretical spectra generated from protein databases.
Trypsin has become the gold standard for protein digestion to peptides for shotgun proteomics. Trypsin is a serine protease. It cleaves proteins into peptides with an average size of 700-1500 daltons, which is in the ideal range for MS (1). It is highly specific, cutting at the carboxyl side of arginine and lysine residues. The C-terminal arginine and lysine peptides are charged, making them detectable by MS. Trypsin is highly active and tolerant of many additives.
Even with these technical features, the use of trypsin in bottom-up proteomics may impose certain limits in the ability to grasp the full proteome, Tightly-folded proteins can resist trypsin digestion. Post-translational modifications (PTMs) present a different challenge for trypsin because glycans often limit trypsin access to cleavage sites, and acetylation makes lysine and arginine residues resistant to trypsin digestion.
To overcome these problems, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated when using these alternative proteases have not been systematically documented.
In a recent reference (2), optimized protocols for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, Lys-C, Lys-N, Asp-N, Glu-C and Arg-C have been created.
Data describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in approximately in 2 days.
- Laskay, U. et al. (2013) Proteome Digestion Specificity Analysis for the Rational Design of Extended Bottom-up and middle-down proteomics experiments. J of Proteome Res. 12, 5558–69.
- Giansanti, P. et. al. (2016) Six alternative protease for mass spectrometry based proteomics beyond trypsin. Nat. Protocols 11, 993–6
Every lab has it—that one thing that is always in short supply no matter how many you order. What is your lab’s “red sharpie”?
If you like this cartoon, there are more like it in our Cartoon Lab.
It began at a sink. Advancing from Dishwasher to Production Manager might seem like an unusual career path, but after speaking with Kris Pearson, the Custom/OEM Production Manager at Promega, it appears perfectly ordinary. I was thrilled to meet with her and discuss both the broad strokes and gritty details of working in Custom/OEM Manufacturing. Continue reading
Not every lab has a tried and true transfection protocol that can be used by all lab members. Few researchers will use the same cell type and same construct to generate data. Many times, a scientist may need to transfect different constructs or even different molecules (e.g., short-interfering RNA [siRNA]) into the same cell line, or test a single construct in different cultured cell lines. One construct could be easily transfected into several different cell lines or a transfection protocol may work for several different constructs. However, some cells like primary cells can be difficult to transfect and some nucleic acids will need to be optimized for successful transfection. Here are some tips that may help you improve your transfection success.
Transfect healthy, actively dividing cells at a consistent cell density. Cells should be at a low passage number and 50–80% confluent when transfected. Using the same cell density reduces variability for replicates. Keep cells Mycoplasma-free to ensure optimal growth.
Transfect using high-quality DNA. Transfection-quality DNA is free from protein, RNA and chemical contamination with an A260/A280 ratio of 1.7–1.9. Prepare purified DNA in sterile water or TE buffer at a final concentration of 0.2–1mg/ml. Continue reading
Fleeing from the scene of a car accident may land your DNA in a local police database in addition to a felony charge. Or it may not. Or it may. The regulation of DNA collection practices varies widely between each state and is the subject of back-and-forth legal decisions, as evidenced by People v. Buza in California.
The humble beginnings of DNA in the courtroom served primarily as confirmatory evidence linking a suspect to the scene of the crime after they were charged based on other evidence. Now detectives are increasingly turning to DNA databases as the first step in their investigations. California alone had 774 matches to the state’s database from cases in May of 2016, or about 25 matches per day. You may have nothing to hide, yet it has become our civic duty to understand how we should use DNA evidence both effectively and ethically in criminal investigations. Is it useful to drop charges for a nonviolent misdemeanor in exchange for a voluntary cheek swab, as being performed in Orange County, CA, or does that take DNA collection practices too far? Should people convicted of misdemeanors be required to give DNA samples? Here we present the current laws in each state and encourage your thoughts below. Continue reading
Antibiotic-resistant bacteria and their potential to cause epidemics with no viable treatment options have been in the news a lot. These “superbugs,” which have acquired genes giving them resistance to common and so-called “last resort” antibiotics, are a huge concern as effective treatment options dwindle. Less attention has been given to an infection that is not just impervious to antibiotics, but is actually enabled by them.
Clostridium difficile Infection (CDI) is one of the most common healthcare-associated infections and a significant global healthcare problem. Clostridium difficile (C. diff), a Gram-positive anaerobic bacterium, is the source of the infection. C. diff spores are very resilient to environmental stressors, such as pH, temperature and even antibiotics, and can be found pretty much everywhere around us, including on most of the food we eat. Ingesting the spores does not usually lead to infection inside the body without also being exposed to antibiotics.
Individuals taking antibiotics are 7-10 times more likely to acquire a CDI. Antibiotics disrupt the normal flora of the intestine, allowing C. diff to compete for resources and flourish. Once exposed to the anaerobic conditions of the human gut, these spores germinate into active cells that embed into the tissue lining the colon. The bacteria are then able to produce the toxins that can cause disease and result in severe damage, or even death. Continue reading
As the July heat and humidity builds, my mind wanders to good wandering places, including natural historical sites close to home.
Don’t get me wrong, there is nothing like a cross-country adventure, but if I drive 700 miles to a family gathering, and cannot converse on the state parks and historical attractions near my adopted home in southern Wisconsin, then what kind of a traveler—or Wisconsin resident for that matter—am I?
You know what I mean. You drive a full day to a family reunion, only to have someone there tell you how they loved a historical attraction minutes from your home, an attraction you’ve never seen—a conversation stopper to be sure.
Case in point: Wisconsin has one of the highest numbers of Native American Indian burial mounds in the US. In case it’s on your way to Wisconsin, our neighbor to the west, Iowa, is home of Effigy Mounds National Monument, part of the National Park Service!
Let’s start with ancient effigy mounds close to home; Here in Madison, Wisconsin, there are mounds on the University of Wisconsin campus, both on Observatory Drive and closer to the Picnic Point path. The web site for the Lakeshore Nature Preserve along Lake Mendota provides some fascinating detail and historical accounts of some of these ancient burial sites, attributed to the Woodland Indians.
Another Independence Day is in the books, and for many of us in the U.S. it included spending time with friends, family, food and the traditional holiday fireworks. Around the world, fireworks add to the enjoyment of many annual celebrations and events. Their colorful visual and audio display has the ability to thrill us, no matter what age we are. Despite growing older I never seem to tire of fireworks; I’ve also noticed that with each passing year the show seems to get more sophisticated. Whether it be a new color or shape or design of firework, pyrotechnic technology seems to improve at an impressive rate.
That got me thinking… how do fireworks actually “work”? Basic chemistry and physics are clearly at play, so in the spirit of a science-related blog I decided to look into this and share what I’ve learned. Continue reading
Forensic lab validations can be intimidating, so Promega Technical Services Support and Validation teams shared these tips for making the process go more smoothly.
- Prepare Your Lab. Make sure all of your all of your instrumentation (CEs, thermal cyclers, 7500s, centrifuges) and tools (pipettes, heat blocks) requiring calibration or maintenance are up to date.
- Start with Fresh Reagents. Ensure you have all required reagents and that they are fresh before beginning your validation. This not only includes the chemistry being validated, but any preprocessing reagents or secondary reagents like, polymer, buffers, TE-4 or H2O.
- Develop a Plan. Before beginning a validation, take the time to create plate maps, calculate required reagent volumes, etc. This up-front planning may take some time initially, but will greatly improve your efficiency during testing.
- Create an Agenda. After a plan is developed, work through that plan and determine how and when samples will be created and run. Creating an agenda will hold you to a schedule for getting the testing done.
- Determine the Number of Samples Needed to Complete Your Validation. Look at your plan and see where samples can be used more than once. The more a sample can be used, the less manipulation done to the sample and the more efficient you become.
- Select the Proper Samples for Your Validation. Samples should include those you know you’ll obtain results with be similar to the ones you’ll most likely be using, and your test samples should contain plenty of heterozygotes. When you are establishing important analysis parameters, like thresholds, poor sample choice may cause more problems and require troubleshooting after the chemistry is brought on-line.
- Perform a Fresh Quantitation of Your Samples. This will ensure the correct dilutions are prepared. Extracts that have been sitting for a long time may have evaporated or contain condensation, resulting in a different concentration than when first quantitated.
- Stay Organized. Keep the data generated in well-organized folders. Validations can contain a lot of samples, and keeping those data organized will help during the interpretation and report writing phase.
- Determine the Questions to Be Answered. While writing the report, determine the questions each study requires to be answered. Determining what specifically is required for each study will prevent you from calculating unnecessary data. Do you need to calculate allele sizes of your reproducibility study samples when you showed precision with your ladder samples?
- Have fun! Remember, validations are not scary when approached in a methodical and logical fashion. You have been chosen to thoroughly test something that everyone in your laboratory will soon be using. Take pride in that responsibility and enjoy it.
Need more information about validation of DNA-typing products in the forensic laboratory? Check out the validation resources on the Promega web site for more information for the steps required to adopt a new product in your laboratory and the recommended steps that can help make your validation efforts less burdensome.