...gene knock-out (left) and targeted DNA knock-in (right) by CRISPR/Cas9. Adenoviral CRISPR vectors (left...left) or CRISPR vectors and donor DNA vectors (right) are transduced to target cells. Within target cells...

...purified by streptavidin high-affinity purification. This system is both highly sensitive and specific ...

...bp, but pay attention to the sequence’s Tm, as a higher Tm give more efficient recombination. We normally... two colonies for simple cloning procedures. Use high fidelity DNA polymerase This will minimize random...

...current period of rapid flux, the chapter might still feel slightly dated by next week.   A stepping stone...

...wanted to think more closely about the wealth of high-throughput sequencing (HTS) data we generate and...the aim of the journal club is to begin building a high level understanding across multiple teams or to ...

...Gamma-retrovirus genome (left) and an HIV lentiviral genome (right.)  HIV seems an unlikely candidate for use as...genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proceedings of the...

...cumbersome to use and lead to lower viral titers due to higher plasmid number. For more information on the key...Lentiviral particles with the VSV-G envelope have high infectivity in a wide range of cell types, so they...

...professionals identify grant opportunities with the highest likelihood of funding and develop a strategy and... project management, and program evaluation are highly transferable. Use your experience and your passion...

...around 2010 and found the scheme very nice at first sight in that Addgene takes care of everything including...research to realize their vision. Their progress might be slow, but if they don't stop, they may be able...

...to Cas9, which requires that each DNA target have high sequence identity to the spacer sequence and contain...to detect single molecules of an RNA species with high specificity (Gootenberg et al., 2017). This system...

...following injection into the bloodstream, but requires a high vector dose and is unable to reach all regions of...small genetic difference between these two mice might drive their divergent phenotypes. Two labs, the ...

...This post was contributed by Kutubuddin Molla, a Fulbright Visiting Scholar at the Pennsylvania State University...Cuttack, Odisha, India. Currently Kutubuddin is a Fulbright Visiting Scholar at the Pennsylvania State University...

..., cells that retain the ccdB gene die off and a higher percentage of transformants contain the ccdB-less...great additions to constructs that are used to drive high protein expression in vivo. Expression plasmids ...

... was to ensure the scientific community obtained high-quality data for our DNA-based reagents. Not only..., one of our first problems was deciding on the right system for us to start using. Prior experimenting...

...Syndrome research, the role of the MECP2 gene, and highlights some of the latest breakthrough therapeutic approaches...

...? Let’s take a look at codon usage and when you might want to optimize codons for expression in a particular... on the codon usage frequency in a reference of highly expressed genes (Sharp and Li, 1987). In 2000 the...

... method, they found that SaCas9 did not display higher levels of off-target activity than SpCas9, confirming...editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge that the...

...positive cells isolated with TREE have a significantly higher frequency of genomic base pair edits of interest...positive cells can be sorted and have a significantly higher frequency of base edits of interest than solely...

...eukaryotic ribosome. One resistance gene on your vector might require two different selection molecules! It all...protein, to visualize vector uptake. These reporters might be used as markers for automated cell sorting or...

...Although AYBE, AXBE, and ACBE do not approach the high product purity of CBEs and ABEs, they could be useful...transversion editors, this editor had relatively high indel rates ranging from 4.7% to 30% at tested sites...

...which help a researcher understand if a material is right for their experiment.     Table 1: Most popular ...learned to clone from the Addgene website.” We delight in seeing our resources shared via social media...

... you are trying to repress a gene, your control might be a system where expression of that gene is knocked...selection of the positive control plasmids should be highly specific to the experiment and the interrogation...

...plasmids expressing variants with optimal function. High-throughput DNA sequencing is then used to measure...mutant strand: A mix of degenerate primers and the high-fidelity Phusion polymerase are used to introduce...

...ionizing radiation, chemotherapeutics, and ultraviolet light. In this post, we will describe the mechanism of...systems, ssODN templates generally provide the highest frequency of HDR edits.  Check out our blog post...

...,000 phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the...intersection of cell signaling, metabolism, and the mighty mitochondrion. You can follow her on Twitter @nieminm...

...financial aspects. Money often carries an emotional weight. Therefore, too much focus on money can dissuade...point, your budget bottom line costs may be rather high. That’s normal, because this is where you can start...

...components of CGBEs. Their eOPTI-CGBE and cOPTI-CGBE had higher editing efficiency and produced fewer indels than...accessible. It's not entirely clear why this is, but it might point to a more dynamic picture of chromatin than...

...LB and weigh pellet. The total weight of each pellet should be ~1-2 g. Pro-Tip Make sure to weigh the empty... plates at 30 ℃ overnight. Plate 2.5 mL of the transformed cells on each of the eight bioassay plates ...are often used for screening, barcoding, or other high throughput multiplexed experiments. These pooled... we recommend the use of a diagnostic digest and high-throughput next generation sequencing (NGS). Select...Workflow Timeline Day 1: Transform, recover, set up overnight growth (Estimated time 2-3 hours) Transformation... Alternatives include Stbl4 cells or other ultra-high efficiency electrocompetent cells that are suitable...of electrocompetent cells is essential to ensure high efficiency uptake of plasmid library DNA. This quantity...

...numbers, 50 or more per cell and have a high copy number. High copy number plasmids should only need to...determine if your plasmid is high or low copy. I didn't get any growth after overnight incubation. What went ...However, a liquid culture is capable of supporting a higher density of bacteria and is used to grow up sufficient... The following protocol is for inoculating an overnight culture of liquid LB with bacteria. Video Watch...Prepare liquid LB. For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle: 4 ...2 mL in a falcon tube, but for larger preps you might want to use as much as a liter of LB in a 2 L Erlenmeyer... sterile aluminum foil or a cap that is not air tight. Incubate bacterial culture at 37°C for 12-18 hr...

...Introduction Many molecular biology techniques require highly purified and concentrated plasmid DNA. This page...microcentrifuge Desktop vortexer Vacuum (optional) Reagents Overnight culture of bacteria transformed with your plasmid...Protocol: Generalized DNA Purification Grow an overnight culture of bacteria . Pro-Tip Refer to appropriate... with DNA preparation. Pro-Tip If your entire overnight culture cannot fit into a single centrifuge tube...of dH 2 O Store Solution III at 4°C Grow 2 mL overnight cultures from single colonies of bacteria containing...often thought that an incubation of 20 min to overnight at -20 °C or -80 °C will improve precipitation... the tube for 5 min at room temperature on the highest setting. Note: You should see clearly separated...