Restriction Enzyme Digestion: Capabilities and Resources

Restriction enzymes recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to these sequences.  These enzymes are the workhorse in many molecular biology applications such as cloning, RFLP, methylation-specific restriction enzyme analysis of DNA, etc.  In order to streamline and shorten these workflows, restrictions enzymes with enhanced capabilities are desirable.

A subset of Promega restriction enzymes offer capabilities that  include rapid digestion of DNA in 15 minutes or less, ability to completely digest DNA directly in the GoTaq® Green Master Mix, and Blue/White Cloning Qualification which allows for rapid, reliable detection of transformants.

To learn more about restriction enzymes and applications, check out Restriction Enzyme Resource on the web. The resource provides everything from information on restriction enzyme biology to practical information on how to use restriction enzymes. This resource also contains useful online tools to help you use enzymes more effectively. It helps you choose the best reaction buffer for double digests, find the commercially available enzyme that cuts your sequence of interest, find compatible ends, and search for specific information on cut site, overhang isoschizomers and neoschizomers by enzyme name.

For added convenience, you can download the mobile app and use the Restriction Enzyme Tool to plan your next digest.

For additional information regarding Restriction Enzyme Digest, reference the supplementary video below.

What Makes a “Good” Buffer?

Buffers are often overlooked and taken for granted by laboratory scientists, until the day comes when a bizarre artifact is observed and its origin is traced to a bad buffer.

The simplest definition of a buffer is a solution that resists changes in hydrogen ion concentration as a result of internal and environmental factors. Buffers essentially maintain pH for a system. The effective buffering range of a buffer is a factor of its pKa, the dissociation constant of the weak acid in the buffering system. Many things, such as changes in temperature or concentration, can affect the pKa of a buffer.

In 1966, Norman Good and colleagues set out to define the best buffers for biochemical systems (1). By 1980, Good and his colleagues identified twenty buffers that set the standard for biological and biochemical research use (2,3).  Good set forth several criteria for the selection of these buffers: Continue reading “What Makes a “Good” Buffer?”

Celebrating Bike-to-Work Week 2019

Every day at the Promega Headquarters in Madison, Wisconsin, many Promega employees trade the crowded Beltline Highway for a scenic route along the bike trails. As our colleagues wind around the lakes and prairies of south-central Wisconsin, they’re reducing greenhouse gas emissions and getting some fun exercise in the process. This week, during National Bike-to-Work week, we’re taking time to recognize our colleagues who opt for a healthier and more sustainable commute. In the video below, hear about how Promega supports our bike commuters from Sam Jackson, an avid biker and Multimedia Specialist at Promega.

Converting RPM to g Force (RCF) and Vice Versa

Radius

g Force or Relative Centrifugal Force (RCF) is the amount of acceleration to be applied to the sample. It depends on the revolutions per minute (RPM) and radius of the rotor, and is relative to the force of Earth’s gravity.

A good, precise protocol for centrifugation instructs you to use the g force rather than RPMs because the rotor size might differ, and g force will be different while the revolutions per minute stay the same. Unfortunately, many protocols are written in hurry and instructions are given in RPMs. Therefore, you have to convert g force (RCF) into revolutions per minute (rpms) and vice versa.

Modern centrifuges have an automatic converter but older ones do not. There is a simple formula to calculate this, but it takes some time to do the calculation. Meanwhile, your cells might die or the biochemical reaction goes on for three times longer than it should.

There are several ways to make conversion:

Continue reading “Converting RPM to g Force (RCF) and Vice Versa”

From BTCI to Africa and Back Again: One Student’s Journey in Science Education

Today’s blog is brought to us by and alumus of Dane County Youth Apprenticeship Program, Aidan Holmes.

In this blog I have the opportunity to write about how my experiences at the BTC Institute as a high school student were instrumental in leading me to my passion for science education, my Peace Corps experience, and my current role as a biotechnology instructor for the very same institute.

I became familiar with the BTC Institute as a student at Marshall High School when our biology teacher organized a biotechnology field trip for us. I loved learning about DNA and biotechnology since 7th grade so attending a field trip like this was an incredible opportunity to engage in hands-on biotechnology. When I learned about the Youth Apprenticeship Program in Biotechnology I knew I had to apply and enrolled during my senior year of high school. Through the program I took a weekly class at the BTC Institute and I worked as a student researcher in a biochemistry lab at UW-Madison. I enrolled for classes at UW-Madison the following year and pursued an undergraduate degree in genetics and a certificate in education and educational services. Continue reading “From BTCI to Africa and Back Again: One Student’s Journey in Science Education”

Colorectal Cancer Awareness Making March About More Than Basketball

In the United States, March means college basketball. “March Madness” brings us the excitement and entertainment of the NCAA college basketball championship tournament. But for a dedicated group of advocates, researchers, patients and families, it means something else entirely. March is colorectal cancer awareness month.

 

According to the American Cancer Society, colorectal cancer will be the third most frequently diagnosed and the second most deadly cancer in the United States in 2019 (1). Most of those who develop colorectal cancer do not have a family history or genetic connection to the disease. However, in some families, cancer occurs more often than expected. A family history of colorectal cancer can suggest a genetic factor. Continue reading “Colorectal Cancer Awareness Making March About More Than Basketball”

Executing a NanoBRET™ Experiment: From Start to Data

This is a guest post from Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics.

“The objective of my experiment was to test the NanoBRET™ assay as if I was a customer, independent of the research and development team which develops the assay.”

Designing and implementing a new assay can be a challenging process with many unexpected troubleshooting steps. We wanted to know what major snags a scientist new to the NanoBRET™ Assay would encounter. To determine this, we reached out to Laurence Delauriere, a senior applications scientist at Promega-France, who had never previously performed a NanoBRET™ assay. Laurence went step-by-step through the experimental process looking at the CRAF-BRAF interaction in multiple cell lines. In an interview, Laurence provided us with some tips and insights from her work implementing the new NanoBRET™ assay.

In a few words, can you explain NanoBRET?
“NanoBRET is used to monitor protein: protein interactions in live cells. It is a bioluminescence resonance energy transfer (BRET) based assay that uses NanoLuc® luciferase as the BRET energy donor and HaloTag® protein labeled with the HaloTag® NanoBRET™ 618 fluorescent ligand as the energy acceptor to measure the interaction of two binding partners.” Continue reading “Executing a NanoBRET™ Experiment: From Start to Data”

Oh, the Places You’ll Go, Thanks to Science!

Today’s guest blog is written by Aparna Shah, a Post-Doc at Johns Hopkins University. Aparna visited the Promega campus in Madison, Wisconsin on January 18, 2019 and offered to share her experiences.


Aparna poses with her artwork at the Promega Employee Art Showcase

I don’t recall ever having won a contest before, let alone the grand prize! In fact, I did a double take when I first read the email informing me that my SciArt submission had been selected as a winning entry for the Promega Art Contest for Creative Scientists. What did I win, you ask? A free trip to Madison, WI to meet with the team behind the contest and explore Promega’s headquarters!

I first heard about the contest on the HelloPhD podcast and considered participating primarily to support the SciArt movement. A couple of days later, I came across a perfectly-timed tweet about the contest that nudged me out of procrastination mode and reminded me to follow through with it. I’m going to take a second here to pitch both HelloPhD and Twitter to you. Regardless of whether you’re an undergraduate student interning in a science lab or a senior postdoc, the HelloPhD podcast is incredible at calming you down while you’re on the roller coaster ride called academia. As for Twitter, I can think of several pros for using it. But in the context of this post, it is one of the best resources for discovering opportunities that match your interests.

Continue reading “Oh, the Places You’ll Go, Thanks to Science!”

Deep in the Jungle Something Is Happening: DNA Sequencing

This blog was written by guest blogger and 2018 Promega Social Media Intern Logan Godfrey.

Only 30 years ago, the polymerase chain reaction (PCR) was used for the first time, allowing the exponential amplification of a specific DNA segment. A small amount of DNA could now be replicated until there was enough of it to study accurately, even allowing sequencing of the amplified DNA. This was a massive breakthrough that produced immediate effects in the fields of forensics and life science research. Since these technologies were first introduced however, the molecular biology research laboratory has been the sole domain of PCR and DNA sequencing.

While an amazing revolution, application of a technology such as DNA sequencing is limited by the size and cost of DNA sequencers, which in turn restricts accessibility. However, recent breakthroughs are allowing DNA sequencing to take place in jungles, the arctic, and even space—giving science the opportunity to reach further, faster than ever before. 

Gideon Erkenswick begins extractions on fecal samples collected from wild tamarins in 2017. Location: The GreenLab, Inkaterra.

Gideon Erkenswick begins extractions on fecal samples collected from wild tamarins in 2017. Location: The GreenLab, Inkaterra. Photo credit: Field Projects International.

The newfound accessibility of DNA sequencing means a marriage between fields of science that were previously largely unacquainted. The disciplines of genomics and wildlife biology/ecology have largely progressed independently. Wildlife biology is practiced in the field through observations and macro-level assessments, and genomics, largely, has developed in a lab setting. Leading the charge in the convergence of wildlife biology and genomics is Field Projects International. 

Continue reading “Deep in the Jungle Something Is Happening: DNA Sequencing”

Microsatellite Instability Symposium at Duke University

On January 23, doctors, scientists and researchers will gather for a symposium about Microsatellite Instability (MSI) at Duke University. During the one-day event, scientists from Duke University and The Ohio State University will share insight into their research on biomarkers, MSI status and GI cancer, Lynch Syndrome, and MSI and DNA mismatch repair deficiency (dMMR).

Continue reading “Microsatellite Instability Symposium at Duke University”