Today’s blog is written by Technical Services Scientist, Joliene Lindholm, PhD.
Many of us have come back to the lab after a summer of field work or a vacation break, but there is usually someone checking in on the lab to make sure the gel electrophoresis box did not completely overflow with dead bugs and the water baths are not completely overrun with exciting new algae. Maybe this was just because I worked in an older building in an entomology department, but why do insects like running buffer so much? Some labs have been completely shut down for months at this point or maybe just a few essential people have been in keeping stocks and colonies going. Some labs have adapted to the new normal and developed guidelines to keep researchers safe while still doing essential work in the lab. See how the Promega Scientific Applications group has maintained this balance.
Headed back to the lab bench? Take some time to make sure you have everything you need to start up your research projects.
Here are a few tips from what I learned in managing a lab after a period of field work to get back into the swing of things:
Today’s blog is guest-written by Susanna Harris, who recently defended her PhD thesis at the University of North Carolina in Chapel Hill.
I just defended my PhD. Nearly six years of blood, sweat, and tears, most of which were cleaned up with Kimwipes while sitting at my desk in a laboratory facing out towards the UNC Chapel Hill football field. Nearly six years of work, all summed up in a handful of slides. Nearly six years of work, explained to my friends, family, and colleagues – a moment I had dreamed of since the fall of 2014.
What I hadn’t dreamed of? That I would be sitting at my small desk in the corner of my room, with no present audience aside from my snoring dogs. That there would be no dinner celebration that carried into a night of fun along Franklin Street. That, unseen by the viewers of my defense, I would be wearing sweatpants as my name changed from Ms. to Dr. Harris.
Pictured: The audience for Susanna’s thesis defense.
Why did I wear sweatpants when I could have worn literally anything in my closet? Because I think it’s hilarious. I believe this situation will end and we will walk away with memories and lessons learned from an extremely difficult time in the history of the world. I want to walk away with one more ridiculous story to add to a long list of “What even was that?” tales from grad school.
Working towards a PhD is hard at any time; let’s not pretend this pandemic isn’t making things even worse. I was fortunate in many ways that my advisor had already moved our laboratory to a new state in 2019, allowing me to adjust to meeting through webcams and working from home before the pandemic changed the lives of all North Carolinians. This has given me a unique perspective to tease out which problems come from distance working and which are the result of Safer-At-Home orders. Based on my experiences, here are a few tips, tricks, and words of warning.
Travel and event restrictions related to the COVID-19 pandemic have caused many scientific conferences to be canceled, delayed or adapted into virtual events. These conferences include the Society of Toxicology (SOT), American Association of Cancer Researchers (AACR), Experimental Biology (EB) and the BioPharmaceutical Emerging Best Practices Association (BEBPA) Bioassay Conference, among many others. For the most up-to-date information, we recommend checking with the hosts of each conference.
These cancellations have disrupted many scientists’ plans to present research, engage with potential collaborators and interact with vendors. At Promega, we’re sensitive to the lost opportunities and are currently exploring potential ways to create these experiences despite so many conferences being canceled.
“We want people to be able to talk directly with us and have
the same warm feeling as a close conversation at a conference, but without
being face to face,” says Allison Suchon, Promega Tradeshow Manager. “We’re
looking at different options to have that same conference feeling but without
the show going on around us.”
To make the most of our time while we build solutions, we
asked Promega scientists for tips on staying connected and informed when you
can’t go to conferences. Here are some ideas we gathered.
Science touches our lives, daily. But far too many scientific concepts and terms are misunderstood and used incorrectly. Even those of us wearing a “scientist” badge sometimes misappropriate terms, which can act to reproduce the misuse.
A basic level of science literacy is so important for all of us. Why? So that when bombarded with comments about vaccination or climate change on a social media site, we are able to sift through the jargon, understand what’s correct and what is not correct, and make decision based in facts vs. internet gossip. With just a bit of knowledge of basic science terms, you are better protected against deception and you’ll know how to sort facts from fiction.
Here are a few general science terms that are commonly misunderstood and misused.
PCR amplification with a proofreading polymerase, like Pfu DNA polymerase, will leave you with a blunt end. However, another thermostable DNA polymerase, like Taq DNA Polymerase, adds a single nucleotide base to the 3’ end of the DNA fragment, usually an adenine, creating an “A” overhang. This “A” overhang can create difficulties when cloning the fragment is your end goal. You might consider creating a blunt end with Klenow or adding restriction sites to the ends of your PCR fragment by designing them in your primers. But why go through all those extra steps, when that “A” overhang allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors? Fewer steps? Who can argue with that?
These assays are relatively easy to understand in principle. Use a primary and secondary reporter vector transiently transfected into your favorite mammalian cell line. The primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. We can use that second marker to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.
While there are many advantages to dual-reporter assays, they require careful planning to avoid common pitfalls. Here’s what you can do to avoid repeating some of the common mistakes we see with new users:
Blue/White colony screening helps you pick only the colonies that have your insert.
Q: Can PCR products generated
with GoTaq DNA Polymerase be used to for T- vector cloning?
A: Yes. GoTaq® DNA Polymerase is a robust formulation of unmodified Taq Polymerase. GoTaq® DNA Polymerase lacks 3’ →5’ exonuclease activity and displays terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerases (including the GoTaq® Flexi and GoTaq® G2 polymerases) will contain A-overhangs which makes them suitable for T-vector cloning with the pGEM®-T (Cat.# A3600), pGEM®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors.
Formal judgement in any context is nerve-racking. Scientists, familiar with being judged, rely on others to evaluate (and hopefully accept) everything from a PhD thesis defense to grant proposals and peer-reviewed journal article submissions. The frustrating part is not knowing exactly what the judges are looking for. Sure there are requirements and guidelines to follow—but how are the judges going to interpret them? It would be a whole lot easier if we could just peek into their minds. Unfortunately for most, that fantasy isn’t likely to turn into reality.
But if you are part of an iGEM team, today is your lucky day! Our own Preeta Guptan volunteers as a judge for the iGEM competition, and in today’s article you will get her insider’s perspective about what iGEM judges look for. You will also get some tips to help you excel in the iGEM competition—and effectively communicate about science in general.
Preeta is an External Innovation Manager at Promega, which means she seeks out and investigates technology that might be valuable for Promega to license or acquire. The opportunity to scout up-and-coming synthetic biology advances was one reason she wanted to be an iGEM judge, but curiosity was at the core of her decision. Preeta and the other judges bring their unique perspectives and experiences to each iGEM project and team they evaluate. Here are some suggestions from Preeta:
Here in Technical Services we often talk with researchers at the beginning of their project about how to carefully design and get started with their experiments. It is exciting when you have selected the Luciferase Reporter Vector(s) that will best suit your needs; you are going to make luminescent cells! But, how do you pick the best way to get the vector into your cells to express the reporter? What transfection reagent/method will work best for your cell type and experiment? Do you want to do transient (short-term) transfections, or are you going to establish a stable cell line?
Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction. Data are presented graphically rather than as bands on a gel.
For those of us well versed in traditional, end-point PCR, wrapping our minds and methods around real-time or quantitative (qPCR) can be challenging. Here at Promega Connections, we are beginning a series of blogs designed to explain how qPCR works, things to consider when setting up and performing qPCR experiments, and what to look for in your results.
First, to get our bearings, let’s contrast traditional end-point PCR with qPCR.
End-Point PCR
qPCR
Visualizes by agarose gel the amplified product AFTER it is produced (the end-point)
Visualizes amplification as it happens (in real time) via a detection instrument
Does not precisely measure the starting DNA or RNA
Measures how many copies of DNA or RNA you started with (quantitative = qPCR)
Less expensive; no special instruments required
More expensive; requires special instrumentation
Basic molecular biology technique
Requires slightly more technical prowess
Quantitative PCR (qPCR) can be used to answer the same experimental questions as traditional end-point PCR: Detecting polymorphisms in DNA, amplifying low-abundance sequences for cloning or analysis, pathogen detection and others. However, the ability to observe amplification in real time and detect the number of copies in the starting material can quantitate gene expression, measure DNA damage, and quantitate viral load in a sample and other applications.
Anytime that you are preforming a reaction where something is copied over and over in an exponential fashion, contaminants are just as likely to be copied as the desired input. Quantitative PCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is paramount for generating qPCR results that you can trust.
Use aerosol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
Wear gloves and change them frequently.
Have designated areas for pre- and post-amplification work.
Use reaction “master mixes” to minimize variability. A master mix is a ready-to-use mixture of your reaction components (excluding primers and sample) that you create for multiple reactions. Because you are pipetting larger volumes to make the reaction master mix, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
Dispense your primers into aliquots to minimize freeze-thaw cycles and the opportunity to introduce contaminants into a primer stock.
These are very basic tips that are common to both end-point and qPCR, but if you get these right, you are off to a good start no matter what your experimental goals are.
If you are looking for more information regarding qPCR, watch this supplementary video below.
