...pleased to report that over 95% of viral capsids in representative viral vector preparations are genome-containing...vector preparations. Addgene has used this method to generally validate our viral vector preparation protocol... people usually report? Addgene and other viral vector manufacturing facilities report the physical titer...precise and more repeatable than qPCR (Taylor et al., 2017). However, because purified AAV preps contain similar...cell transduction assays. Wild-type AAV2 has been reported to have a near-perfect physical-to-infectious ... However, for recombinant AAV2, the same study reported a physical-to-infectious particle ratio of 50:...al., 2010). The specific infectivity of viral preparations is defined by the ratio of physical viral particles...

...General protein expression  m15 pREP4*, ** Kanamycin (pREP4) Cis-repression of the E. coli T5 promoter (found... (lac repressor on the pREP4 plasmid). Expression vector cannot have p15A origin of replication F-, Φ80ΔlacM15...as M15(pREP4) use a lac repressor to act directly on the expression plasmid in order to repress transcription...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysS...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysE...Tetracycline RecA-deficient; best for plasmids with repetative sequences.  F- ompT lon hsdSB (rB- mB-) gal ...toxic or insoluble proteins Origami2 (DE3)** Streptomycin and Tetracycline Contains highly active thioredoxin...

... offering virion preparations to the scientific community. Addgene's AAV preparations have worked well...lentiviral preps for the delivery of Cas9 endonuclease, Cas9 nickase, Cas9 activators, Cas9 repressors, select...money producing virus from select vectors in the repository yourself, you can now order ready-to-use virus...lentivirus (with many CRISPR tools included among the preps that are currently available) and adeno associated...with optogenetic tools coming soon). The viral preparations undergo rigorous quality control testing at ...faster by producing, titering, and testing viral preps for them. At the same time, we’re developing ourselves...scientists and accelerate research." What viral preps are available now? There are a wide variety of tools...

... own machinery to replicate the viral genome and proteins. This is, for the intrepid researcher looking...that in a bit! Viral genomes and replication When it comes to replication, viruses tend to march to the ...methods of replication. In fact, an entire classification scheme, known as the Baltimore replication classes...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit: ...an intermediary DNA replication step between DNA and mRNA. DNA genome replication can involve an RNA or...before being translated into proteins. RNA viruses replicate their genomes using a variety of RNA or DNA intermediaries... bacteria and eukaryotes, which use a binary replication process whereby one cell copies itself and “splits...

...streak for single colonies, sub-culture, and prep with a DNA prep kit or your lab's favorite in-house protocol...plasmid yields from your prep, try our tips below! How to optimize your plasmid prep We'll discuss the following...for plasmids with repeat elements, such as viral plasmids with inverted terminal repeats. We use NEB Stable...plasmid DNA and lead to degradation of your prep. Many plasmid prep kits recommend performing an extra wash...How to Get Better Plasmid Midiprep Yields BitesizeBio: Better Plasmid Midipreps Part II: What Causes Low...updated on March 21, 2018. Most of the time, plasmid prepping is a breeze. You get your stab from Addgene, streak... multiple techniques for a particularly stubborn prep. Check the bacterial strain Increase the volume...

... the 3’ UTR of the reporter mRNA via an MS2 coat protein (NLS-MS2-RFP) the reporter mRNA, which contains...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...proteins to allow translation to occur. For a TRICK reporter mRNA this displacement leads to a change in the...GFP protein that binds to the coding region of a reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) an RFP...translation, both GFP and RFP are bound to the reporter, causing it to appear yellow. The act of translation...of the mRNA, leaving only a red FP bound to the reporter. Because it contains a nuclear localization signal...copies of the FP-coat protein fusions to their reporter mRNAs. Using translation inhibitors cycloheximide...

...trans-acting RepA protein under the control of IPTG. Because RepA is a plasmid replication initiator protein...genes closer to the origin of replication will be overrepresented compared to those further away. As a...increases, the amount of repressor protein also increases thereby intensifying repression of the gene of interest...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes...regulation by using a promoter that is responsive to a repressor protein, the increase in expression due to copy...Segall-Shapiro et al., they chose a TALE protein as the repressor because TALEs can be programmed to bind tightly...sequences and have been shown to achieve ~100 fold repression in E. coli. In addition, TALEs bind non-cooperatively...

...taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...an —NH2 (amine) group from the target base and replaced it with a double-bonded oxygen. This simple enzymatic...calling it the “edited strand.” Uracil excision repair leads to the first base transversion editor The...most CBEs include an inhibitor of base excision repair (BER), the cellular process that catches mutated...creating an “abasic,” or empty, DNA site, and then replacing the incorrect base with a G. Pro tip! Abasic sites...even better cytosine transversion editors. New DNA repair proteins fused to CGBEs Instead of using UNG, the...fusions of a CBE with other BER components: the DNA repair protein rXRCC1, or the DNA binding and lyase domain...

...mismatch repair (MMR) system. MMR’s job is to correct DNA mismatches that occur during DNA replication. There... and repressor CI. For recombineering purposes, a temperature sensitive version of the repressor gene,...lowering the temperature allows the repressor to re-nature and again repress expression of exo, beta and gam...Overview of using Lambda Red recombineering system to replace a gene of interest with an antibiotic resistance...as non-selectable DNA fragments, such as gene replacements and tags.  The typical frequency of recombinants...targeted, it’s best to determine the direction of replication through your region of interest and design an...it’s easier to not determine the direction of replication and instead design oligos that target both strands...

...DNA, they grow and replicate, and then their DNA is harvested, much like one would prep an individual plasmid...things: underrepresented plasmids (plasmids that were lost or “dropped out”) and overrepresented plasmids...of 0.5 representing perfect uniformity (see Figure 3A). Addgene finds that this representation is most...for screening experiments, so the scale of the DNA prep is larger. And since the library contains many plasmids...library amplification and sequencing. Maintain representation of the library during transformation First,... to ensure all components of the library are represented. Typically, Addgene uses electrocompetent Stbl4...off the plates and pelleted for DNA extraction. Prepare library product for next-generation sequencing ...

...causes the breaks) and host cell DNA repair factors (which repair those breaks) makes Cas9 extremely effective...inactivating genes), while breaks repaired by homology directed repair (HDR) pathways can modify the sequence...which sequence replacement holds some kind of fitness advantage but error-prone repair of the gene would...causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining (NHEJ) pathway...low. Understanding the biological cause of this repair bias is a fascinating (and yet unanswered) question...approach to improve the efficiency of sequence replacement by HDR. Understanding Cas9 biophysics enhances...studies) to boost the efficiency of sequence replacement in vivo. We discovered that single stranded donor...

...a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein...protein, the repressor protein is removed from the DNA. With the repressor protein absent, transcription is...promoter to induce transcription of the phage repressor cI. Repressor cI inhibits transcription from phage promoter...expression of a reporter gene. When light is present, YFI is inactive, preventing repressor cI synthesis ...with this post, check out our follow up post on repressible promoters. How are inducible promoters regulated...promoter. This promoter requires removal of the lac repressor (lacI protein) for transcription to be activated...presence of lactose or lactose analog IPTG, the lac repressor undergoes a conformational change that removes...

...AAV and lentiviral preps we have released in the last few months: Lentiviral preps for expressing SARS-CoV...lentiviral preps to speed up COVID-19 research even further. The following lentiviral preps are now available...2-N-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 N protein  pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro...pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a Lentiviral preps for expressing ...pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro) and NSP2 (pLVX-EF1alpha-SARS-CoV-2-nsp2-2xStrep-IRES-Puro) are also...dependent Cre and FLPo And more!  New lentiviral preps SARS-CoV-2 protein expression The Krogan lab quickly... pooled library is now available as a lentiviral prep. It's a human CRISPR inhibition library that inhibits...

...phase oocytes, thus allowing repair to take place before DNA replication. Indeed, they observed that 41... were able to distinguish between repair from the template vs repair from the maternal MYBPC3 allele. ...indicating that repair proceeded through NHEJ rather than HDR. Figure 1: Homology-directed repair in targeted...Note: After this blog was published, a bioRxiv preprint that questions the conclusion of inter-homologue...Nature gives us even more to talk about, as it represents another step towards CRISPR germline editing ...different gRNAs, and electroporated them with an ssODN repair template in induced pluripotent stem cells made... efficiency of only 27.9% and homology-directed repair (HDR) taking place in 41.2% of edited cells. The...

...and providing lentiviral preps of select lentiviral vectors from the repository for you. We perform test... gRNA representation, produce high titer lentivirus from the library, ensure gRNA representation in the...particular organism available at Addgene. Percentages represent the fraction of that total with the indicated ...excited to provide ready-to-use CRISPR lentiviral preps to researchers across the globe. Why provide CRISPR...in science, getting a high titer Cas9 lentivirus prep takes a bit of time and effort that, for many groups... protocol only to find that the low titer of her prep makes it unuseable. With the large size of the SpCas9...test infections with all of our viral preps and host our expression data (western blots or fluorescent ...

...have added the prep to your cart and checked out.  Prices for Packaged on Request viral preps will be available...Request prep and run the same quality control assays we run with all of our in-stock viral preps: ddPCR...on Request prep 6–9 weeks after placing your order.  Can I order Packaged on Request preps through a distributor...AAV composed of an AAV plasmid in the Addgene repository and their choice of five different serotypes.... and ordering a Packaged on Request viral vector prep.    Excited? So are we! This expansion will ...ordering high-quality materials from the Addgene repository. Read on to learn more.  What will be available...available?  Over 3000 AAV plasmids in the Addgene repository will be available for Packaged on Request service...

...has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged... The researchers took a FRET reporter and adapted it into a BRET reporter by using NanoLuc® Luciferase...and NanoLuc® Luciferase are available from the repository. We encourage people to go to Addgene to get ...couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...host cells. The fluorescent moiety of the BRET reporter was used to sort successfully infected/expressing...NanoLuc luciferase. Both GpNLuc and OgNLuc BRET reporters produced bright signals without exogenous excitation...near 600 nm – a clear demonstration of why a BRET reporter can improve deep tissue imaging. The constructs...

... to cleave and activate a reporter. The detectable signal from this reporter can be measured and quantified...and a Csm6 specific reporter (in the same fluorescent channel as the Cas13 reporter), they could significantly...method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK and SHERLOCKv2) (Gootenberg... termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen et al., 2018). Both SHERLOCK and... SHERLOCK- Specific High Sensitivity Enzymatic Reporter UnLOCKING Cas13 (C2c2) was first identified in...molecules. In SHERLOCK a quenched fluorescent ssRNA reporter is added to the reaction. Cleavage of the quenchable...amplification step in combination with the ssRNA reporter enables SHERLOCK to detect DNA or RNA with atomolar...

...activators and repressors to target genes. Repressing target genes using dCas9-based repressors Early experiments...sufficient to repress transcription by blocking its initiation. In mammalian cells, robust repression requires...transcriptional repressors to the promoter region of the gene of interest. dCas9-based repressors are useful...researchers to knock out, knock in, base edit, activate, repress, and even image your favorite gene. With so many...array of Cas9s (many available through Addgene’s repository) and make it easy to find your perfect match..... Image from David Liu.   Activation and repression using Cas9 Don’t want to permanently modify the...particular gene isn’t feasible. For example, temporary repression or knockdown allow you to study essential genes...

...recombination. Yeast Replicating plasmids (YRp):  These vectors contain an Autonomously Replicating Sequence (ARS...mammalian vectors, will catch you up on the core replication and resistance features of yeast vectors and ...includes but is not limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic...homologous recombination to facilitate simple gene replacement/mutation. Yeast expression plasmids used in the...vectors must contain a yeast-specific origin of replication (ORI) and a means of selection in yeast cells...antibiotic selection markers.  The yeast origin of replication (Please note: This first section primarily pertains...ve also noted some features required for the replication of fission yeast, Schizosaccharomyces pombe, ...