...in a sample. Digital PCR assays for mutation screening Now that we have introduced how digital PCR works...indel mutations resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome ...the bi-allelic mutations typically desired when screening clones for complete functional knockout of your...interested in harnessing the power of digital PCR screening to validate your genome edits, this paper provides...Remember that although digital PCR is a great screening tool, it still requires target DNA sequencing ...PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells. PLoS ONE 11, e0153901–...

...your rate of interactions when screening! Tips for two-hybrid screening Minimizing false positives: Run... these discoveries to create a new system for screening protein-protein interactions. In later years, ...reporter gene cloned downstream of a promoter containing the target sequence of the DBD. The DBD-POI fusion...protein interact and enable intein splicing (5). Screening with yeast two hybrid systems While initial studies...plasmid transformants into a diploid strain for screening), or arrayed on a 96 well plate where each well...a defined prey fusion.  The former allows for screening greater numbers, but the latter eliminates the...the step of identifying prey proteins after screening. It is also possible to screen libraries of bait ...

...should acquire during their PhD and postdoctoral training. Addgene executive director Joanne Kamens has ...scientific society in your field. My leadership training began with the NPA where I gained invaluable experience...function without long distance communication and learning to work with people despite the distance improved...leadership institute’ weekends would entail intense training to develop our leadership and management skills...junior leaders what I had experienced and learnt. Opening doors to further opportunities  Having gained leadership... students to think about their career options, gaining transferable skills such as leadership skills can...

...two major repair pathways – Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) – and... pathways work, as this could be relevant when planning your experiment. Here, we’ll discuss NHEJ, and...mediated DSBs in the genome. Non-homologous end joining steps Unlike HDR, NHEJ is active throughout the...nucleases (Artemis), and structure-specific end cleaning enzymes (Aprataxin, Tdp2) that function to make... breaks, and could result in deletion of the intervening DNA (“pop-out” deletions) if NHEJ joins the distal...repair (“pop-in” insertion) provided a template containing compatible overhangs is available. Cas9 can also...David Wyatt is a graduate student interested in determining how the structure of broken ends impacts how ...

...such as new training programs (like at the Keck Graduate Institute), funding for training (such as the...speaking out and writing about issues in our science training infrastructure for some years.  He spoke eloquently... to choose institutions with allies engaged in training excellence to begin effecting change.  Panelists...advisors only know about academia and alternate training needs to be provided to take advantage of all ...the conference organizing team.  The topics were Training, Structure of the Workforce, Metrics and Incentives...Scientists must do science to be happy so the beginnings of solutions can’t come too soon.  Watch the ...

...another great option for mammalian cloning, and is geared towards cloning multi-TU expression constructs ...blog Plasmids 101: Golden Gate Cloning High Complexity Golden Gate Cloning with NEB’s DAD New MoClo basics...Modular cloning is a popular DNA assembly tool used to build single- and multi-gene constructs for a ...goals are more general in nature, or you aren’t planning on using K. phaffii, then the MoClo YTK Kit specifically...should also take a look at the Yeast GoldenBraid Cloning System (Pérez-González et al., 2017). In addition... kit is a great starter for your general plant cloning experiments. Addgene also has two additional MoClo... kit is also compatible with the Joint Modular Cloning Kit which it builds upon (its ‘basics’ MoClo plant...

...transferable” skills you have developed in addition to learning how to be a scientist. Why not stack the deck ...skills in ways that will also enhance your science training and that will put you in position to pursue a ...develop those transferable skills while you are also learning to be an excellent scientist. Leadership and management...association) Collaboration and teamwork One of the defining characteristics of nonacademic science careers... science training...

...I understand? How does the information I’ll be learning relate to me as an individual and Addgene as a...constantly update our processes and get new ideas from learning experiences like workshops at Lab Central. Because...

...As the saying goes, “failing to plan is planning to fail.” Preparation is a key step to any experiment...

...efficiency (Koblan et al., 2018). Adenine base editors The beginning of adenine base editing Nicole Gaudelli... David LIu’s lab created an adenine base editor that would convert adenine to inosine, resulting in an...Creating an adenine base editor requires an additional step because there are no known DNA adenine deaminases...cytosine and adenine base editing Grünewald et al., 2020 SPACE Has both cytosine and adenine base editing...homology-directed repair (HDR). Compared to non-homologous end joining, HDR occurs at a relatively low frequency, and ...base editors: cytosine base editors (CBEs) and adenine base editors (ABEs).  (There are also RNA base ...editors from postdocs Alexis Komor and the first adenine base editors from Nicole Gaudelli in David Liu’...

...multiplexed CRISPR systems for high-throughput screening are vastly useful for mapping the combinatorial...ligation steps to build barcoded lentiviral plasmids containing one or more gRNAs. As each ligation reaction ...digestion and ligation result in plasmid libraries containing combinations of different gRNAs in each plasmid... lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12 lentiviral...library, but this should be verified by NGS before beginning your experiments. With CombiGEM and CRISPR platforms...Hiroko, et al. "Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide...Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87...

...development through thoughtful planning and honest feedback DePace’s yearly planning meetings revolve around ...various issues, the PI is responsible for determining meaningful solutions. PI-trainee communication builds...setting and career planning help alleviate the anxiety of graduate and postdoc training. On the other side...goals.      Although myIDP is great for career planning, it shouldn’t be used in isolation. Without clear... her lab (pictured above). DePace holds yearly planning meetings with each trainee to create a supportive...to facilitate honest conversation. Goals and Planning Worksheet: Lists accomplishments, research/professional...Starting with a recap of accomplishments, from learning new techniques to finally figuring out a tough...

...themselves. Therefore, your cloning strategy needs to identify cells containing the plasmid construct you...positive selection, negative selection, and/or screening. We’re focusing on positive and negative selection...After transformation, plate the cells on medium containing the corresponding antibiotic. For example, if...cassette, you would plate the cells on kanamycin-containing agar. Only cells carrying plasmids that contain...with or without your inserted DNA) and further screening will help you determine if the plasmid backbone...Auxotrophy and prototrophy come into play during cloning if you’ve designed your experiment using an auxotrophic...amino acid). Many common E. coli strains used for cloning are auxotrophic for specific amino acids. When ...

...Hughes Medical Institute. Are you currently on or planning to go on the academic job market? In addition ...-10 minutes to present your research vision uninterrupted and then you will begin working through your...overview and address the aims in more depth in the remaining time. The typical chalk talk structure is: What...

... to help you build a great poster.  Tips for designing your poster Check conference guidelines To begin...you should always follow conference rules when designing your poster, since they can differ. 48” x 36” ...

...deep mutational scanning.     3 reasons to be interested in deep mutational scanning Hundreds of thousands...Scientists use deep mutational scanning to simultaneously test how multiple amino acid changes affect... to phenotype that can be used with mutational scanning: ligand binding assays, phage display assays, ...Several methods can be used to create mutational scanning libraries. While each of these methods works well...fragments in a single-tube reaction. For mutational scanning experiments, libraries of DNA fragments that contain...assembly is better suited for creating small, focused scanning libraries. PFunkel: Of prior existing comprehensive...Variants on a Large Scale Using Deep Mutational Scanning.” Nature Protocols 9.9 (2014): 2267–2284. PubMed...

...Plasmids 101: TOPO Cloning Plasmids 101: Golden Gate Cloning Read all of our Plasmid Cloning Posts Resources...more recently developed Gibson Assembly Cloning and Gateway® cloning technologies, as well as several others...powerful cloning tool Molecular biologists first saw the potential of this system for enhancing cloning efficiency... prep plasmids containing the ccdB gene, which can then be used for downstream cloning applications. While...strains for more strain info. Additional cloning resources As cloning continues to evolve, understanding all...Addgene.org Choose a Molecular Cloning Technique Find Plasmid Cloning Protocols Check Out All of Our ...If you’re into cloning, you’re probably aware that there are several methodologies currently available...

...Cervical cancer screening in Ghana Gladys is currently working to enhance cervical cancer screening in Ghana....cervical cancer screening for a limited group of Ghanaian women by including PCR screening for HR HPV alongside...tests are commonly used in Ghana, there is little screening for DNA markers like the presence of high risk...tests currently in use. Gladys will use plasmids containing a variety of HPV genes as positive controls in...by these screens can then undergo more routine screening allowing for early cervical cancer detection, ...her in the future. Future awardees We will be opening the Addgene/Seeding Labs Plasmid Grant to a third... cancer screening...

...If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true...starting point: Ligation-independent cloning Ligation-independent cloning (LIC) was first developed in the... doing large amounts of cloning. Assembly is scarless, unlike Gateway cloning, and the method’s flexibility...at Addgene Ligation-Independent Cloning Choosing a Molecular Cloning Technique Choosing Your Perfect ...the 1990s. While traditional restriction enzyme cloning used short sticky ends, LIC employed the exonuclease...bacteria after transformation. LIC is a reliable cloning method, but it is limited by its sequence constraints...specially-designed plasmids. Sequence- and ligation-independent cloning: A SLIC makeover In 2007, LIC received an important...