...DNA elution choices: TE, Tris buffer, or water Figure 2: Tris-EDTA, Tris, or water? The buffer you...protocols that recommend eluting in water, Tris-EDTA (TE), just Tris buffer, or some other variations. Does... affect your downstream experiment. TE (10 mM Tris-HCl,1 mM EDTA, pH 8.0) buffer is the best buffer...the stability of your preparation for a long time. Tris buffer controls the pH, while the EDTA chelates ... enzymatic reactions, a valid alternative can be Tris buffer (with no EDTA) or water. These are two of... DNA through their kit. The advantage of using a Tris buffer is that it allows better control of the pH...for a short period, and there are indications that Tris buffer is better than water in these conditions....

...When cloning by restriction digest and ligation, you use restriction enzymes to cut open a plasmid (backbone... of how to do perform restriction digests.   Overview of the restriction cloning process. Both ...backbone will contain a variety of restriction enzyme cut sites (restriction sites) downstream of the promoter...DNA (insert) that has been cut by compatible restriction enzymes. An enzyme, DNA ligase, then covalently...sequence of interest (green, insert) are cut with restriction enzymes to generate compatible overhangs that...backbone covalent.  Before beginning the restriction digest and ligation process, you should carefully...these both must have compatible cut sites for restriction enzymes that allow your insert to be placed into...

...Thirteen: Guest Contributors of 2023 8 Great Depositor Research Stories Interested in contributing to the Addgene...bloggers and subject matter experts who have contributed to our blog in 2024. Thank you to…   Marty LaFleur...Kits We are very grateful for each and every contributor to the blog. Thank you! More resources on the...

...chromosome and for stringently controlled plasmids. Binding of IHF (green) redistributes DnaA (red) from ...chromosomal DNA.  Plasmids are said to be under stringent control when they depend on the host cell’s initiation...consider the replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...appropriate time—for example, the cell must have enough nutrients available to complete the entire round of replication...assists with the process: it bends the DNA to redistribute the DnaA from high-affinity boxes to low-affinity...replication. A variety of other proteins also contribute to this regulation in different ways: methylating...DNA replication and regulation Plasmids must also strike a balance: replicating too slowly will eventually...

...email [email protected] if you would like to contribute to the catalog. The Catalog Will Start With Neuroscience...antibodies available at launch were created by James Trimmer, PhD, of the University of California Davis School... Figure 1: Antibody against PSD-95 from the Trimmer Lab. Information about the antibody, the target...Nguyen K, van der List D, Murray KD, Engebrecht J, Trimmer JS (2019) A toolbox of IgG subclass-switched recombinant...

...you to all who have contributed to the Addgene blog this year! Your contributions have helped researchers...: Erik Lee SnappSharing is Caring, But How to Distribute Open Hardware? Author: Benedict Diederich Course-Based...

...Have you ever tried digesting with XbaI or ClaI restriction enzymes and gotten unusual or unexpected ...methylation may affect your restriction digests. Why methylate? Restriction enoducleases are so ubiquitous...It turns out that restriction enzymes are one half of naturally occurring restriction modification systems... from the restriction modification systems, yet can still affect whether certain restriction enzymes can...associated with any restriction modification systems, their sequences may overlap with restriction sites, inhibiting...sequences to prevent them from being degraded by restriction endonucleases. A given prokaryote typically has... has genes encoding one or a few restriction modification systems containing methyltransferases that add...

... Ingredient Concentration or % Purpose Tris HCl 62.5 mM Buffer SDS 2% (w/v) Detergent...buffer such as PBS or TBST. Stringent stripping The stringent stripping solution uses heat and detergent...Biosystems offer pre-made stripping solutions or kits. Mild stripping The mild stripping solution uses low pH...lose by trying to strip and reprobe. In this case, we recommend using mild stripping solution (see below...with the antibodies, especially with more stringent stripping methods. If you are planning to detect multiple...later, strip it thoroughly first. Then continue on with your western blot as normal! Stripping your membrane... to strip blots, and the best choice will depend on your specific experiment. Remember, stripping your...

...from the bioelectricity field In addition to sharing tools to study bioelectricity, bioelectric researchers...Developmental Bioelectricity symposium’s “What is Bioelectricity?” Journal of Bioelectricity ...of biology known as bioelectricity is revealing the important role of bioelectrical signaling mediated ...first introduced to bioelectricity when I attended the Developmental Bioelectricity satellite symposium... these slower-changing bioelectric states in living cells. Since bioelectrical signals are controlled ...Michael Levin. "The body electric 2.0: recent advances in developmental bioelectricity for regenerative and...  All living cells with a membrane possess an electrical state. This state, known as the resting potential...

...code, find and fix security-critical bugs, and contribute to constant updates of the software. In hardware...community to regularly replicate designs worldwide. Distributing hardware via globally established makerspace...

... technologies distributed through Addgene over our first 10 years. 10 most distributed plasmid technologies...technology, but pSV-PGC1 was Addgene's first distributed plasmid and was created in the lab of my thesis...Bruce Spiegelman. It was also the only plasmid distributed in our 1st year, 2004, and it still gets requested...Plasmids Shipped! - By 2010, Addgene had hit our stride. We shipped our 100,000th plasmid. A variety of...up with all the new plasmids. So far, we have distributed over 10,000 CRISPR/Cas9 plasmids to more than...

...Researchers in developing countries often find themselves on the front lines of emerging global challenges...Seeding Labs to better serve those in developing countries. Developing an international scientific community...community Seeding Labs, a nonprofit founded in 2007, strives to expand the opportunities and scientific resources...resources available to researchers in developing countries--because scientific talent is everywhere, but ...around the world. As part of our own mission, we strive to help build the international scientific community... are always taking steps to increase plasmid distribution and disseminate knowledge around the globe. .... Addgene’s progress in international distribution As you can see from the chart below, we are making ...

...of how to analyze a restriction digest:     Restriction digest tips and tricks: The following tips ...plasmids. Here, we'll cover restriction digest analysis. Diagnostic restriction digest Diagnostic digests...Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction enzymes...to confirm the sequence of all the plasmids we distribute. This method is time-intensive, so we recommend...organization of different features within the plasmid. Restriction analysis can also be used successfully even if...relative sizes of the resulting DNA fragments. Restriction digests are commonly used to confirm the presence...backbone. To do this, you'll use enzymes with restriction sites that flank the insert. You will need to...

...providing plasmids to researchers in developing countries. We are proud to introduce the 5 scientists who...molecular biotechnology for improving expression of nutritionally and therapeutically significant proteins. In...Encoded H2O2 Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging” ACS Chemical...Expression Systems Learn about Global Plasmid Distribution ...

...alteration) Expensive buffers Tris-tricine 2.5–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours...Limited protein size range Tris-acetate 40–500 kDa Tris-tricine 150 V, 1–3 hours Good for ...Running conditions Pros Cons Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and...that Bis and Tris gels run proteins in different patterns, due to their differing chemistries. You therefore...-life; can alter proteins due to high pH Bis-tris 6–400 kDa MES for proteins <40 kDa 180 V, ... protein alteration) Expensive buffers Bis-tris 6–400 kDa MOPS for proteins > 30 kDA 200 V...therefore cannot compare Bis and Tris gels or blots to each other. Once you know your gel type, you’ll need...

...project We will ship you  2 µg of DNA in 20 µl of Tris buffer suitable for molecular biology experiments...this period, we’d love to hear from you if you’ve tried out this service! Let us know what you think. You...excited to begin offering this service to further contribute to our mission to “accelerate research and discovery...

...complex and dynamic field. We’re going to focus on a tried and true method of finding enhancers by isolating...mono-methylated and acetylated histones, but not tri-methylated histones, functions as an enhancer. We... me1, me3, and ac – denoting mono-methylation, tri-methylation, and aceylation, respectively (these ...suppose you have a piece string that’s 10 meters long. You twist and roll the string into a tight ball. Now...This post was contributed by guest blogger, Aneesh Karve, CTO at Quilt Data. This post was originally...follows: (mono_methylation + acetylation) - tri_methylation In the next section we’ll apply the above formula...Now the two ends of the string, instead of being 10 meters apart, are mere millimeters apart. Similarly...

... an exciting and emotional time!” George Church tries to recruit people who are nice as top criterion ...latest results as soon as the gels ran.” Dr. Ellis tries to lead his team as a scientists and a friend. George...

...and potent liver transduction. While we have not tried it, it might be worth investigating for both Q6 ...cross-react with each other. Multiple labs have tried to transfer PHP.eB's enhanced CNS tropism to other...understanding that AAV-BR1 has the same genome size restrictions as other AAV. Therefore, one should aim to keep...

...end up stressed and rushing, no matter how hard I tried to build in extra time before the experiment. What...any experiment. That, more than anything else I tried, made a huge difference in my ability to focus, ...used Roberson Lab, UPenn. Western blotting. Retrieved Feb. 7, 2022. https://www.med.upenn.edu/robertsonlab...

...modular code. Use tried-and-true tools We use Python, Django, Apache, and MariaDB. Using tried-and-true tools...tracked. Terminology and language are tricky Names for things will trip everyone up at some point, decide ...messiness of biology. These examples are intentionally trivial, but they're here to help tell this story. In this...plasmid and now it’s time for our lab technicians to retrieve the sample and get it ready for shipping. In the...contents might leave you sad and confused when a tricky procedure failed and one of your techs started ...

...biology. While not everything is open-access, I have tried to prioritize open-access resources whenever possible...

...plasmids for transfer to recipient cells during a tri-parental mating scheme. Mobile-CRISPRi integrates...transferred to a recipient bacteria strain of interest by tri-parental mating. Donor bacteria contain a chromosomal...presence of trimethoprim, cultures treated with a folA gRNA were more sensitive to trimethoprim when compared... which encodes the target for the antibiotic trimethoprim. When Enterobacter aerogenes, Klebsiella pneumoniae... the folA gene product not been a target for trimethoprim, there would be no changes in sensitivity. Pooled...

...and lead to you being remembered as a person who tries to find solutions that benefit everyone. Even if...negotiation, I will view the “winner” positively if they tried to understand my position and to find a creative...

...have dedicated science teachers. I tried contacting our school district’s science curriculum coordinator ...the technology in an unethical manner, but I also tried not to be an alarmist.  The overarching guideline...and science communicator, and I strive to be a volunteer and contribute to my community. This past year...all of these roles by reaching out to my school district not just as a dad, but as a scientist. When our...it. PTAs have more flexibility than the school district itself in terms of what they do with their discretionary...like this without them! If you live in a school district that has a lot of parents in STEM careers, chances...chances are they’re wondering how they could contribute to the school, too. Though you can recruit parents...

...month ago. Why haven’t we received our order? I tried searching my PO in the track order portal and nothing... Ordering genes on plasmids online is a little trickier -- but at Addgene, we try to make the process ...does not own the material in our repository. We distribute it on behalf of depositing institutions. Addgene...

... Paolo Colombi Should I store my plasmid in TE, Tris, or water? And what about temperature? Find the ...

...for efficient recombination? A3: The most we've tried to insert so far has been 1kb. We used homology ...template, you could then: Confirm your HR by doing Restriction Fragment Length Polymorphism (RFLP) (see Figure...

...the grant or your PI may edit everything you’ve tried to draft, but you’ll still be practicing and getting...

...is really important. For my Postdoc position, I tried to read at least 50 papers on the topic before I...This guest blog post was contributed by Seán Mac Fhearraigh, PhD, of www.phdadvice.com. I sold out, well...

...molecules are sensitive to blue light. Many have tried to make green molecules, but then alongside blue...Optogenetics is a toolset that lets you turn the electrical activity of neurons on or off using pulses of...neuron or set of neurons, you can see how it contributes to behavior or pathology, and by disabling neurons...deafness, or other conditions. Addgene: You've contributed various plasmids to Addgene over the years, many...popular. Why has it been so important to you to distribute these tools as freely as possible? Boyden: I ...for different ions. Right now, we can modulate electricity. If you could selectively let in calcium or some...brain. Addgene: Do you have any special tips or tricks for someone looking to try these optogenetic tools...

... Maguire and colleagues, other researchers have tried to modify it to improve yields. For example, transfecting...for isolating exo-AAVs rely on differential centrifugation steps to separate cell culture supernatant ...

...started learning proper cell culture technique, I tried to find an app that could help me with calculations...information in the app is restricted to the NEB inventory only, the restriction enzyme sequence resource...This post was contributed by guest blogger Sean D. Stacey. Opinions on the apps discussed in the article...comes in handy when I use protocols that require restriction enzymes or PCR. The app contains a double digest...digest tool that is only helpful for NEB restriction enzymes since the instructions use NEB buffers. Nonetheless...name to discover all the information about that restriction enzyme or share it via email with a colleague...colleague. The app is also great if you’re adding a restriction site to a primer, you only need to skim the alphabet...

...tools are abundant! In this post, we will review tried-and-true protein expression control tools as well...

...marker at non-physiological levels. Scientists have tried varied approaches to combat these issues. One method...maintained when grown on media containing the missing nutrient. Scientists can exploit these host mutations by...auxotrophy. When grown on media NOT containing the nutrient, the host cells will die unless they have incorporated...  Marcy Patrick...

...these elusive teachers, supporters and advisors? I tried to start a more formal mentoring program at my company...They all found the support of the group to be intrinsic to their success. Their stories made it clear ...Goal setting is easier with more perspectives contributing to the process Senior people (of which there...

...most widely known of these products is TRIzol® (also called TRI Reagent®, RNAzol®, QIAzol®  and sold by...homogenate and shaken. After a quick centrifugation (not ultracentrifugation!), the layers of phenol and chloroform...After homogenization in TRIzol®, insoluble material is removed via centrifugation and the supernatant is... loaded onto cesium-chloride gradients and ultracentrifuged for up to 20 hours to separate the intact ...have, access to one or more large, expensive ultracentrifuge.  Figure 1: An outline of the different...out to develop a protocol that skipped the ultracentrifugation altogether. Chomczynski and Sacchi showed... by many different companies). TRIzol® is an all-in-one acid-guanidinium-phenol solution that combines...

...writing three years ago on a field trip in Indonesia. Since then, I have tried many things: photography, making...This post was contributed by guest blogger Sarah Schmidt, a Marie Curie Fellow at The Sainsbury Laboratory...hosted by Nature), and Addgene are open to guest contributions. If you decide to start your own blog, check...

...confused, bored, or particularly excited about? If you tried tweaking your pitch throughout the conference, what...

...answer should come soon, but not instantly. Addgene tries to get back to customers within 24 hours, and we...time at Addgene, I’m confident that these tips and tricks will apply to any biology-related customer support...

...’t tell if the bags contained flora or fauna. I tried to find out what certain ingredients might be used...very welcoming team from China’s Ribobio, the illustrious speaker list attracted a host of sponsors based...and depositors. China is in one of the top 10 countries in number of plasmid requests from Addgene. ...

...keywords close enough... If in the past you have tried to combine multiple terms in an Addgene plasmid ...plasmids from the James Trimmer Lab published in eLife. Addgene is currently distributing 94 plasmids expressing...Availability to Industry: Restrict search to plasmids which can be distributed to industry research scientists... GCaMP containing plasmids are available for distribution. If your experiment involves delivering a calcium...

...answers come sooner rather than later. Once you’ve tried it, let us know how xCas9 is working for you in ...another method to create point mutations, also has a strict editing window about 15 nt upstream of the PAM....included continuous outflow of phage, increasing stringency and promoting survival of only the broadest-targeting...

... brain tumor biology. When not snipping DNA, he tries random things (101 lessons on Spanish, C++, guitar...DEMONSTRATE GENOME EDITING BY THIS TOOL This post was contributed by guest blogger Pooran Dewari. Any views in ...performs Sanger sequencing on select regions of all distributed plasmids as part of quality control, but does...with the NgAgo system indicates that it is very tricky to use and fussy to optimize. One reason could ...inconvenient. Another possible reason for the trickiness of NgAgo is the inability of mature NgAgo protein...enzyme." DNA-scissors (blog). Blog website. 4. Hsu, Patrick D., Eric S. Lander, and Feng Zhang. "Development...

...cells to let the virions out. We recommend using a Tris-based lysis buffer and sonication.  The obtained...density gradient centrifugation (e.g., iodixanol) using a high-speed ultracentrifuge. During the spin,..., and the two helper plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure 1:...ddPCRAAV Purification by Iodixanol Gradient UltracentrifugationAAV Production in HEK293 Cells...

... be a shortage of amusing reports where someone tries to “optimize” a particular standard lab protocol...technical knowledge and judgement. For particularly tricky cases, team discussions can often lead to otherwise...

...     Over the last decade, my peers and I have tried to move away from calling non-academic careers and...hands-on lab opportunities. You can get your hands in nitrile gloves again! The opportunities are there once ...

...If you’ve heard of containerization, but haven’t tried it or are confused by the concept, don’t worry–you... developed for a singular purpose (ex. adapter trimming, quality score analysis, k-mer counting, or sequence...first n items. To be sure, we went through much trial and error to learn the scripting language, but our...process. For example, our bbduk process created trimmed FASTQ files as output, which we specified as input...

... glycine pH 2.5 and immediately neutralized with Tris pH 8-8.5. Low pH glycine is very effective at disrupting...and blue triangles. 2) An antibody immobilized on a bead binds specifically to the blue triangle protein...not the other. 3) The beads are collected by centrifugation or a magnet. 4) Unbound proteins are washed...target protein. The beads can be collected by centrifugation (agarose beads) or a magnet (magnetic beads...particularly advantageous because they do not require centrifugation between washes which can cause loss of beads...containing non-ionic detergents, such as NP-40 or triton-X, to preserve native protein conformations and...proteins, including those in the nucleus, more stringent buffers may be needed. Since many factors, including...