Targeted Gene Modification in Prairie Voles Using CRISPR and pGEM®-T Easy Vectors

As the number of children diagnosed with autism spectrum disorder (ASD) continues to rise, the search for a cause continues. Scientists have been studying genetically modified oxytocin receptors, which have shown promise as a target for studying ASD-related behaviors. One of the obstacles to designing robust scientific experiments for investigating potential ASD causes or treatments is the lack of a truly appropriate model organism for social behaviors in humans (1). Sure, there are the traditional lab rats and lab mice that demonstrate a certain level of social behaviors. However, there has been a loss of natural social behaviors in common lab mice strains because of the reduction in genetic complexity from inbreeding and adaptation to captivity (2). These animals cannot fully represent the depth of human social behaviors, including the ability of humans to form lasting social bonds (1).

Enter: The prairie vole (Microtus ochrogaster).

Continue reading “Targeted Gene Modification in Prairie Voles Using CRISPR and pGEM®-T Easy Vectors”

Reliable DNA Purification from 3D Cell Cultures

Traditionally, scientists have relied on flat, two-dimensional cell cultures grown on substrates such as tissue culture polystyrene (TCPS) to study cellular physiology. These models are simple and cost-effective to culture and process. Within the last decade, however, three-dimensional (3D) cell cultures have become increasingly popular because they are more physiologically relevant and better represent in vivo conditions.

A spheroid of ~1,000 human liver cells. Image provided by Insphero.
Continue reading “Reliable DNA Purification from 3D Cell Cultures”

T-Vector Cloning: Questions, Answers and Tips

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.

Continue reading “T-Vector Cloning: Questions, Answers and Tips”

The Future of Synthetic Biology: A Recap of iGEM 2019

After attending the iGEM Giant Jamboree last year and being completely blown away by the projects presented (check out this article or this one), I didn’t think I’d be as astonished this year. I attributed part of the awe I felt over the caliber and quality of the projects to my wide-eyed naiveté, having never attended the event before. The second time around, the “first-time” novelty long worn off, I didn’t expect to feel that same level of amazement.

I couldn’t have been more wrong.

After three days of impressive presentations, I once again felt that same astonishment as I prepared to watch the presentations of the 6 finalists. With good reason—the projects presented by the six finalists completely blew my mind!

Continue reading “The Future of Synthetic Biology: A Recap of iGEM 2019”

How should I evaluate DNA isolated from FFPE samples to ensure success?

Part two of three. You can read part 1 here.

Formalin-Fixed Paraffin embedded (FFPE) samples are being used in increasing numbers of molecular assays. In my last blog I discussed some of the pre-analytical variables that can affect results obtained when using FFPE samples. Laboratories can increase the quality of downstream results by controlling variables where possible. While exacting control over the sample acquisition and fixation process can improve results, quality testing of incoming samples is a crucial step in assuring optimal results. There are numerous methods that can be used to evaluate the quality of samples and they can provide different information that can be used to assess sample integrity and suitability for different applications.

Continue reading “How should I evaluate DNA isolated from FFPE samples to ensure success?”

A Diamond in the Rough: New Applications of Diamond Nucleic Acid Dye

Diamond™ Nucleic Acid Dye (Cat# H1181) is a safe, inexpensive and sensitive fluorescent dye option that binds to single-stranded and double-stranded DNA and RNA. Diamond™ Dye typically is used for staining electrophoresis gels to visualize nucleic acids in a similar to its carcinogenic counterpart, ethidium bromide. However Diamond™ Dye has several advantages: gels stained with Diamond™ Dye can be visualized using either UV or blue-light transilluminators. Also, a wash step after staining is not necessary when using Diamond™ Dye, unlike what is typically recommended for ethidium bromide.

Besides staining electrophoresis gels, there are other applications for this diamond in the rough. Highlighted below are two fascinating uses of this multifaceted tool: touch DNA localization and qPCR detection.

Continue reading “A Diamond in the Rough: New Applications of Diamond Nucleic Acid Dye”

Discussing the Future of Gene Editing at CRISPRcon Midwest

crisprcon_banner
Walking in to the first session at CRISPRcon Midwest.

Last week, a diverse group of stakeholders attended CRISPRcon Midwest, hosted by the Keystone Policy Center and the University of Wisconsin–Madison. The goal of the day-long conference was to emphasize the importance and value of gene editing technology, and how it must be communicated deliberately between scientists, the public, policymakers, and other stakeholders.

Julie Shapiro, Senior Policy Director of Keystone Policy Center, acted as Emcee for the event. Given the diverse group of attendees, she mentioned in her opening remarks that the event organizers were “seeking conversation, not consensus” and emphasized the “power of respectful dialogue.” A slide overhead showcased the ground rules for the day, which included statements such as “dare to listen, dare to share, and dare to disagree.”

crisprcon_poll_word_cloud
Word cloud generated from live polling results at CRISPRcon Midwest.

CRISPRcon aimed to included voices beyond those represented by keynote speakers and panelists, so they incorporated live polling through an online app to keep the audience engaged and an active participant in the conversations throughout the day. From the opening remarks, it was clear that this conference would not just deliver on its promise of thoughtful conversation about the science, but build further understanding about the societal impacts of a rapidly advancing technology.

Continue reading “Discussing the Future of Gene Editing at CRISPRcon Midwest”

Nucleic Acid from FFPE Samples: Effects of Pre-Analytical Factors on Downstream Success

Part one of three

Peer-reviewed publications containing data dervived from analysis of nucleic acids isolated from FFPE samples have increased dramatically since 2006.

Formalin Fixed Paraffin Embedded samples (FFPE) have been a mainstay of the pathology lab for over 100 years. Initially FFPE blocks were sectioned, stained with simple dyes and used for studying morphology, but now a variety of biomolecules can be analyzed in these samples. Over the past 10 years we have discovered that there is a treasure trove of genomics data waiting to be unearthed in FFPE tissue. While viral RNAs and miRNA were some of the first molecules found to be present and accessible for analysis starting in the 1990s, improvements to DNA and RNA extraction methods have demonstrated that PCR, qPCR, SNP genotyping, Exome and WGS are possible. This has resulted scientific publications of DNA and RNA data generated from FFPE samples starting in 2006, and today we see immense amounts of data generated from FFPE—with nearly 2000 citations in 2018 reporting sequencing of FFPE samples.

Depending on the type of project, prospective or retrospective, the genomics scientist has an opportunity to affect the probability of success by better understanding the fixation process. The challenge with FFPE is the host of variables that have the potential to negatively affect downstream assays.

Continue reading “Nucleic Acid from FFPE Samples: Effects of Pre-Analytical Factors on Downstream Success”

A Quick Method for A Tailing PCR Products

Some thermostable DNA polymerases, including Taq, add a single nucleotide base extension to the 3′ end of amplified DNA fragments. These polymerases usually add an adenine, leaving an “A” overhang. There are several approaches to overcome the cloning difficulties presented by the presence of A overhangs on PCR products. One method involves treating the product with Klenow to create a blunt-ended fragment for subcloning. Another choice is to add restriction sites to the ends of your PCR fragments. You can do this by incorporating the desired restriction sites into the PCR primers. After amplification, the PCR product is digested and subcloned into the cloning vector. Take care when using this method, as not all restriction enzymes efficiently cleave at the ends of DNA fragments, and you may not be able to use every restriction enzyme you desire. There is some useful information about cutting with restriction sites close to the end of linear fragments in the Restriction Enzyme Resource Guide. Also, some restriction enzymes require extra bases outside the recognition site, adding further expense to the PCR primers as well as risk of priming to unrelated sequences in the genome.

Continue reading “A Quick Method for A Tailing PCR Products”

Curiosity and Collaboration: A PhD Journey

Concepcion Sanchez-Cid didn’t know she wanted to be a scientist when she was older. She grew up with a love of music and played the violin, but her curiosity and eagerness to learn drove her down the path for a career in biomedical research.

Hear more of Concepcion’s story:

 

As a Master’s student at the University of Granada, Concepcion studied biotechnology and landed an internship at the Promega Europe Training and Application Lab (PETAL) in France. She worked with the Applications Team to develop protocols for DNA and RNA extraction from soil. When she decided to pursue a PhD, she received a sponsorship from Promega and enrolled as a student at the University of Lyon while also remaining an employee at PETAL.

Concepcion says that the balance between both worlds—academia and industry—provide her with technical skills and a unique support network that has helped shape her PhD thesis work. “Working at a university and a company at the same time…you get very different feedback from people that are very specialized, and they really know what they’re doing, so at the end you integrate everything,” she says. “It’s one of the things I appreciate most about my PhD.” Continue reading “Curiosity and Collaboration: A PhD Journey”