...fold (Davis et al., 2002). However, polybrene is highly sensitive to freeze-thaw cycles and should be stored...resistance marker expression following transfection, higher-level expression after approximately 72 hours is...

...1948 as a variation of the more commonly observed light emission by fluorescence. The widespread use of ...distances that are too miniscule for conventional light microscopy. Don't FRET - Use these FRET resources...

...are a multitude of known AAV serotypes that have slight variations in their viral capsids. Novel forms ...AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the human population. These pre-existing...

...of large-scale assembly useful, you may ask? You might be surprised by how multi-purpose it can be. Let... Plant Parts Kit Resources on the Addgene blog High Complexity Golden Gate Assembly with NEB’s DAD Interview...

...expression can change as well. The more frequently and tightly bound DNA is to histones, the more compacted it...occurs at CpG islands (regions of the DNA with a high frequency of C and G nucleotides). The conversion...

...gradually. I didn’t become Chair of a Committee overnight (however this can be done if you already have ...was involved with postdoctoral issues first at Brigham and Women’s Hospital/Harvard Medical School and...

...my experience the best set-up is panelists in a slight semi-circle (so they can see each other) on comfortable...redirect the conversation. The person you cut off might be miffed but the other 200 audience members will...

...think I need. Unlike lentivirus, AAV requires a higher number of viral particles for efficient gene transfer...only harvest the cell pellet. My yield would be higher if I used PEG precipitation, but for my experiments...

...cluster of enhancers. CAPTURE 2.0’s multiplexing and high resolution allowed the Xu Lab to simultaneously ... chromatin's dynamic role in gene regulation The high resolution and multiplexed chromatin ‘photos’ generated...

...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...

...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 ...

...? 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...

... 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...

...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...

...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...

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

... 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...

...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...

...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...

...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...

...least 30 min. The high pressure will prevent your gel mix from boiling over at high temperature. Pro-Tip...your agar is at the right temperature, we recommend using a laser thermometer. Light the flame at the plate...them out at room temperature overnight to allow them to dry. After overnight drying, we place the plates...the appropriate liquid solvent. See table to the right for appropriate antibiotic concentrations. Antibiotic...its cap or aluminum foil (but do not make an air-tight seal!) and tape the bottle with autoclave tape. ... from the autoclave and will cool your gel-mix slightly. Even so, you should always use thermally insulated...resistant to the antibiotic. Incubate both plates overnight at the appropriate growth temperature and check...

...protein, Apolipoprotein L3 (red) overnight, blocked, and incubated overnight with Anti-Desmin [D7]. Plates...protocol may need to be adjusted to accommodate slight differences between products. Addgene does not ...plate seal and incubate at 37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare the...set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody incubation...set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 4: Secondary antibody incubation...set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 5: TMB reaction Carefully remove...to blue and generally takes between 15 to 30 min. High concentrations of HRP yield a greenish solution....

...Protocol Version 1.0. December 2006. Copyright Addgene 2006, All Rights Reserved. This protocol is provided...ligation, use only long-wavelength UV light. Short wavelength UV light will increase the chance of damaging...culture plate. Incubate cells at 37°C, 5% CO 2 overnight. Although cells should regularly be passaged in... in ten 6 cm plates and grow at 37°C, 5% CO 2 overnight. Day 2: b. The target cells should be approximately...Plate target cells and incubate at 37°C, 5% CO 2 overnight. Day 2: b. Target cells should be approximately...plate, add between 0.05-1 mL virus (add 0.5 mL for a high MOI, and 0.1 mL for a low MOI). Scale the amount...cell line. d. Incubate cells at 37°C, 5% CO 2 overnight. Day 3: e. Change to fresh media 24 hours after...

...PEI μg ratios provided high transfection efficiencies as measured by the highest proportion of GFP positive...without limiting cell growth. Left panels: bright field images; right panels: GFP channel images. Last reviewed...meter Stir plate Magnetic Stir Bar Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and ...the packaging cell line is critical for obtaining high viral titer. 293T cells should be split 3 times ...purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmids should also be...transfection to determine what ratio gives the highest percentage of GFP positive cells. Refer to the ...

...Casting tray Well combs Voltage source Gel box UV light source Microwave Reagents TAE ( recipe here ) Agarose...you to visualize the DNA under ultraviolet (UV) light. Caution EtBr is a known mutagen. Wear a lab coat...gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density... in the first place. Carefully load a molecular weight ladder into the first lane of the gel. Note: When...the second stop and carefully raise the pipette straight out of the buffer. Carefully load your samples...destain for 5 mins. Using any device that has UV light, visualize your DNA fragments. The fragments of ...minimize damage to the DNA. Note: When using UV light, protect your skin by wearing safety goggles or ...

...are having trouble getting colonies, you might want to use higher competency cells. Additionally, if your...amplify your insert DNA. It is important to use a high fidelity taq polymerase to minimize mutations. The...the digest goes at least 4 hours and as long as overnight. If you are going to use only one restriction ...final product is going to be very large (>10kb) you might want to use electro-competent cells instead of the...insert plate that are not correct. If you have a high number of colonies on your recipient plasmid alone...more colonies you will need to pick) and grow overnight cultures for DNA purification. After purifying... by Sequencing PCR based cloning carries a much higher risk for mutation than restriction enzyme based...

...controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine ...alternative such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5...health of the cell line is critical for obtaining high levels of virus. HEK293T cells should be split 3...purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmid should also be ...transfection to determine what ratio gives the highest percentage of GFP positive cells. Refer to the ...pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high transfection efficiencies as can be seen here by...green fluorescent protein expression (green in the right panels) with a limited effect on cell growth. Last...

...are having trouble getting colonies, you might want to use higher competency cells. Additionally, if your... on your gene of interest (YGOI for short). You might need to express YGOI in cultured mammalian cells...the digest go at least 4 hours and as long as overnight. If you are going to use only one restriction ...final product is going to be very large (>10kb) you might want to use electro-competent cells instead of the...insert plate that are not correct. If you have a high number of colonies on your recipient plasmid alone...more colonies you will need to pick) and grow overnight cultures for DNA purification. After purifying...

... homepage! You can track the number of plasmids right below the search bar and more stats further down...

...a: That will warm up the entire lab (hint: you might need some mini marshmallows…). We’ll choose 4 submissions...

...! Our mascot who has supported us over the past eight years now has a new friend to join them. Blugene...

...starting their careers in the lab and even those who might just need a refresher on some best practices for...