...generating long overhangs which allows the single stranded regions with homology to anneal a polymerase, ...hour or so, the sample is immediately ready to transform into competent cells. The master mix of enzymes...Gibson assembly master mix for 1 hour, and then transform into competent cells. The success rate of this...if the ends of the fragments have stable single stranded DNA secondary structure, such as a hairpin or ...would directly compete with the required single-stranded annealing and priming of neighboring assembly ...

...strong transcriptional terminator BBa_B1002 (from IGEM) upstream of the MCS to reduce transcriptional readthrough...rely on exogenous enzymes, require multiple transformation and plasmid curing steps, and can generate ...worked too. Rhamnose induction relies on the transcriptional activator rhaS. To allow manipulation of bacteria...Anirudh S. Channarasappa, and Howard M. Salis. "Translation rate is controlled by coupled trade-offs between...

...across samples and equal protein transfer from the gel to the membrane. A loading control is critical for...identically, used to assess naturally occurring random biological variability. If one were studying the...purple) and nonspecifically through the Fc region (orange). The Preimmune serum control will not bind to .... Always probe the loading control on the same membrane that the target protein was measured on.    Flow...

...the department? Are they successful at getting grants and funding? Beware of bullying Your mental health...careers?    Will you be encouraged to develop transferable skills by attending career events or being involved...your aspiration? Will you help or at least observe grant writing? Get equipment quotes? Understand administrative...consider less expensive, portable areas of research (transgenic mice—out, zebra fish—in) Team attitude vs. dog...resources on the Addgene blog Check out our transferable skills guide Learn about careers in science ...

..., and effective for wide range of mycoplasma. These methods cannot guarantee complete removal of mycoplasma...the media after autoclaving hence, there is no assurance of the serum being contamination free. The aerosols...commonly infecting mycoplasmas are used. A broad range of primers encompasses almost all the infecting ...filters, for media filtration. There is no 100% assurance that this process can avoid contamination but ...

...collaborates with NIH-funded researchers and utilizes grants to support our own initiatives, Addgene is deeply...developments. These abrupt changes threaten to slow transformative biological discoveries and life-saving medical...archiving their biological materials during funding transitions. Enabling resource sharing: Our operating model...services can be listed as direct costs from a CRO in grant applications, addressing the new funding structures...

...Find the anti-CRISPR plasmids Tagging human transcription factors with GFP Kevin White’s lab recently ...deposited a set of CRISPR plasmids to tag human transcription factors with GFP. As part of the Encyclopedia...these donor plasmids will help researchers map transcription factor localization and binding via ChiP-seq...ChiP-seq. Learn more about ENCODE Find the transcription factor tagging plasmids If you have a new CRISPR...

...and packaging plasmids as well as transfer plasmids. Download the orange lentivirus background.         ...our fluorescent protein background. Download the orange fluorescent protein background.           Did...px or 1080 px).         Do you do a lot of transformations with the plasmids you receive from Addgene?...

...PGC-1a1, a transcriptional coactivator induced in skeletal muscle by exercise and endurance, protects ...have reported a mechanism based on studies of transgenic mice that helps to explain how it works. The ... skeletal muscle expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into...

...weight by SDS-PAGE, and are subsequently transferred to a membrane. A primary antibody recognizing the protein...is added, and will bind to that protein on the membrane. Then, addition of a secondary antibody allows...showing GFP expression in cells that have been transfected with a plasmid encoding GFP. Actin, a common ...bind to proteins on the cell surface, or the cell membrane can be permeabilized to allow antibodies to bind...

.... Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection by Alyssa Cecchetelli...editing. Using a fusion between Cas9 and reverse transcriptase and a prime editing guide RNA, it can introduce...

...fluorescent tags in neurons. Their toolkit named ORANGE (Open Resource for the Application of Neuronal ...in neurons.  Read the paper in bioRxiv Find the ORANGE toolkit   Cas9/gRNA ribonucleoprotein delivery...delivery by lentiviral capsid-based bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages...for packaging Cas9/gRNA ribonucleoproteins for transient expression. They replaced the tetraloop of the...

...at Addgene, and send random sets of 4 people out to lunch.   I think these random lunches would be a wonderful...-make-time-for-that” type projects. Cross-team “Random Lunches” With more employees, Addgene has naturally...Kamens, responded by starting something we call “Random Lunches.” Once or twice a year, she’ll mix up the...

... tools for diverse applications. For example, RANbodies can be used as an alternative to conventional ...sdAbs were used to create GFP scaffolds for transcriptional activation.  sdAbs in the clinic sdAbs have...also been some interesting work done developing transgenic mice that express camelid variable domains (aka...Llamas Required - Synthetic Nanobodies Against Membrane Proteins More resources from addgene.org  Plasmids...

...sequences to sequences in a database ExPASy translation tool: Translates a DNA sequence using all six open reading...Annotates features, BLASTs, aligns, digests, and translates a sequence.   NEBCutter2: For finding restriction...information, such as expression patterns, post-translational modifications and polymorphisms Codon optimization...

...scientists and technology transfer offices trust us to work for them with full transparency. We don’t have to ...resources continuously raising money from donations or grants to fulfill their mission. Addgene is fortunate ...Natashia, Nicole, Olivia, Pamela, Racheal, Ramsey, Randall, Ray, Rebecca, Renee, Shannon, Susanna, Tenisha...

...B) Strategy for leak-free transcriptional control. C) Iterative transduction workflow overview. Image ... by integrating a SL-insensitive fluorophore (LSSmOrange) as an internal control and a SL-sensitive fluorophore...fluorescent biosensor has a high sensitivity (50 nM-500nM range) and a high specificity for SLs in vitro, and they...

...). G. stearothermophilus grows at temperatures ranging from 30-74 °C, with an optimal growth temperature...or G) Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - 45 °C 50 - 70 °C Genome...were incubated in reaction buffer at temperatures ranging from 25-80 °C. Activity is expressed as Kcleave...Harrington et al tested GeoCas9’s editing skills by transfecting HEK293 cells with GeoCas9 or SpCas9 RNP’s and...

...in the cell. gDNA guides will also need to be transfected into the cells of interest. In contrast, RNA ...must be expressed from a plasmid or in vitro transcribed, and changes in gRNA secondary structure can ...removal RNA-guided CRISPR enzymes cause double-stranded breaks, with either blunt (Cas9) or staggered ...Cpf1) cleavage. NgAgo takes a different approach, randomly removing 1-20 nucleotides from the cleavage site...

... sensor AAV GABA is the primary inhibitory neurotransmitter in vertebrates, but methods for directly imaging...GABA sensor (pAAV.hSyn-FLEX.iGABASnFR) has good membrane localization and brightness. The F102G variant... through imaging. Since it’s illuminated by red/orange light, Archon1 can also be used in conjunction ...pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2]) of this membrane-embedding voltage detector. Find Archon1 and its...