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...A few months ago, we shared an introduction to immunofluorescence (IF) - a common method for visualizing...of interest within a cell or tissue. In that introduction, we broke down the method into six general steps...improve antibody binding for some targets, they can destroy it for others. That said, organic solvents’ impact... K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction to Performing Immunofluorescence Staining. Methods...resources on the Addgene blog: Antibodies 101: Introduction to Immunofluorescence Antibodies 101: Epitope...Epitope Availability Antibodies 101: Introduction to Antibodies Resources on Addgene.org Find recombinant...

...capsids (i.e. the part of the virus that determines tropism) have been developed that increase transduction...expressing plasmids is generated. Diversity is introduced into the cap plasmid library by inserting short...which, in the case of Chan et al, was neurons and astrocytes. Why screen for IV deliverable AAVs? Typically... AAV vectors expressing RGB proteins under the control of an inducer, i.e. tet-off transactivator (tTA...expression in the CNS and PNS Previously to broadly introduce a transgene into a specific cell type in the brain...fluorescent tag-then-express system that allows for controlled induction of multi-color labeling of neurons ...

...researchers developed viral vector systems based on retroviruses like Moloney murine leukemia virus (MMLV). The...system that could do both, Addgene depositor Didier Trono and collaborators turned to the lentivirus HIV-1...plasmid contained a viral envelope to dictate the tropism, or types of cells the vector could infect. Finally...vectors are incredibly popular in the lab - the Trono lab’s lentiviral packaging and envelope plasmids...Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. Science. 1996 Apr 12;272(5259):263-7. PubMed ...Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. J Virol. 1998 Nov;72(11):8463-71. ...

...resistance is swift, as few as five years after introduction. Current antibiotic strategies are nonspecific.... Phage therapy fell out of fashion with the introduction of antibiotics, but the field is currently undergoing...antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating into the bacterial ...lytic phage-resistant. When the lytic phage is introduced, it targets only the bacteria that are antibiotic...gain resistance to a lytic phage through the introduction of a crRNA (shown in blue). Infection with the...Bacterial Genome Engineering Treating Muscular Dystrophy with CRISPR Gene Editing Using CRISPR to Edit ...

...unable to reach all regions of the CNS. PHP.B, a neurotropic variant of AAV9, is better than AAV9 at gaining...receptor used by PHP.B viruses. Creating PHP.B, a neurotropic AAV9 variant PHP.B’s specificity for C57BL/6J...CNS transduction and transduces the majority of astrocytes and neurons across multiple CNS regions. PHP....Ly6a seemed the most likely candidate due to its strong linkage to the PHP.B phenotype and its expression...mice strain specific Ly6a SNPs on PHP.B binding in vitro. They found that when LY6A contained C57BL/6J SNPs...PMID: 30819613. Hordeaux, Juliette, et al. "The neurotropic properties of AAV-PHP. B are limited to C57BL...

...! The team used other molecular techniques to introduce time modulation of the TEV cleavage and improve...dCas-KRAB activator and the dCas-VPR repressor are introduced into the hPSC genome using AAV1 and contain an...Find the plasmids at Addgene! CRISPR RNP electroporation and AAV donor infection (CRISPR-READI) enables...new tool for synthetic biology uses CRISPRi to control gene expression. This includes a CRISPRi-based ...affinity), as well as GRAB_NEmut as a negative control. Find the sensors at Addgene! New yellow fluorescence...AAV5 and AAVrg. Find these AAVs at Addgene New controls, biosensor, chemogenetic and optogenetic AAV for...

... it is essential to include proper controls. As a positive control, include a sample that is known to ...This method also requires more optimization and controls when developing, as secondary antibodies can also..., purified protein, or a peptide. The positive control will confirm that the procedure is working and ...and should always be included.  For a negative control, include a sample that does not contain the target...where the target is not expressed. The negative control will help you determine if there is non-specific...

...size using electrophoresis, typically capillary electrophoresis. Capillary electrophoresis reads a fluorescent...process, see our blog post on NGS plasmid quality control. NGS is generally cheaper if you have many samples...to sequence our packaged viral DNA for quality control. This gives us a faster turnaround time than short-read...Validation: seqWell Addgene’s Tips for Plasmid Quality Control Additional Resources on addgene.org Molecular...

...New compact, efficient prime editors By Susanna Stroik The Liu lab recently used phage-assisted continuous...limitations and enable impressive editing efficiencies in vitro and in vivo. Incorporating reverse transcriptase... vectors, CreVLPs, and eVLPs for in vivo and in vitro applications.   Figure 2: Modular features... enzyme, Lee and Cheah et al. achieved precise control over when and where the enzyme is active, drastically..., then the other, a dual-enrichment and mass spectrometry workflow identifies proteins bearing both labels...light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells. Nature Methods...

...most as cancer cells are amenable to culture in vitro. Virologists too have benefited from the ability... ‘Master’ stock, which can be stored in liquid nitrogen long term, preferably some of these vials will...will be stored in a separate backup liquid nitrogen store to prevent loss from equipment malfunctions. One...also monitor the stock levels you have in liquid nitrogen for your Working and Master stocks of each cell... laboratories to carry our independent quality control for a range of testing, from animal doping to forensics...Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture Basics Guide Abcam Cell Culture ...

...are available to confer greater transcriptional control, assist with proper protein folding, and deal with...Contains mutated lac permease whch allows for linear control of expression F- ompT lon hsdSB (rB- mB-) gal ...polymerase (RNAP) from the bacterial genome under control of the lac repressor, which is inducible by the...cases, some strains carry an additional measure of control such as the pLys plasmid, which suppresses basal...difference being that the latter provides tighter control of basal expression. What if I don't see protein...sources, so fear not—there are a few simple troubleshooting measures that can help get you back on track...

...Emerging Investigators Podcast breakdown Intro Chapter 1 - Introduction to Mark Springel, Olivia Ho-Shing, ... In this episode of the Addgene Podcast, we introduce you to the Journal of Emerging Investigators, an...developing one-on-one mentorship programs. Chapter 6 and Outro - Interview with high school student researchers...basic plasmid biology Use or CRISPR 101 eBook to introduce students to this amazing genome enegineering technology...

...Yeast Prototrophy Kit from Markus Ralser's Lab -  Use this kit to complement unused auxotrophies in Saccharomyces...to a quick (~5 min) Hot Plasmids podcast that introduces you to 4 new plasmid technologies from one of... Plasmid 2: Magneto 2.0 form Ali Guler's Lab - Control neuronal activity with a magnetic field. Listen...strains.  Listen to Our Segment on The Yeast Prototrophy Kit Special thanks to our sound engineer/producer...

..., such as the introduction of heterozygous endogenous epitope tags, or the introduction of dominant mutations... examples from their lab include using AAV to introduce epitope tags into the endogenous alleles of the...2008). They have also used AAV gene editing to introduce naturally occurring cancer-causing mutations into...However, while CRISPR is remarkably effective for introducing non-sequence specific frameshift mutations through...efficiency of AAV-based gene editing via the introduction of a double strand break by using a targeted...

...coverage, mutational efficiency, control over where mutations are introduced, and scalability (see Supplementary...favoring of certain types of mutations, difficulty controlling the rate of mutagenesis, and low throughput. ...mutagenesis PCR that uses mutagenic primers to introduce all desired codon changes (step 1 in the image... high-fidelity Phusion polymerase are used to introduce point mutations into the newly synthesized DNA...Error-Prone PCR”. Methods in Molecular Biology In Vitro Mutagenesis Protocols, pg. 103-109. PubMed PMID:...

...Shine-Delgarno sequences used in this so-called bicistronic design (BCD). The 49 vectors in this kit can ...of varying strengths centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013...terminators can be used to precisely tune your polycistronic E. coli expression constructs. Some applications... Living Bacteria Using an Orthogonal Ribosome-Controlled Fluorescent Reporter."ACS chemical biology 11.1...vector that enables protein expression in any in vitro translation system. The gene set was carefully chosen...been used to produced recombinant proteins for in-vitro assays: Koch, Daniel, et al. "A pull-down procedure...

... the strength of the signal in comparison to a control sample. Southern blotting applications range from...causes sickle cell anemia. The mutation happens to destroy a sequence recognized by the restriction enzyme...gels and next-generation sequencing techniques Introduced in 1977, Sanger sequencing, also known as chain-terminating...referred to as sequence DNA as it synthesized by electronically recording fluorescence activity as fluorescently...differences in ion flow caused by the different electrochemical properties of nucleotides (8). Compared to ...sequences among DNA fragments separated by gel electrophoresis." Journal of molecular biology98.3 (1975): ...

... competent cells (protocol) Preparation of electrocompetent cells (protocol) At Addgene, we use the...competent cells (protocol)  Transformation of electrocompetent cells (protocol)  Cells prepared using ...with ampicillin resistance.  After a plasmid is introduced to bacteria, the bacteria is typically grown ...competent cells, don’t forget to do a negative control (plate your cells without adding plasmid) to make...have contamination.  Also conduct a positive control (test a plasmid that transforms well in other competent...

...substrate generates a proportionate luminescent signal, strong enough to detect from proteins expressed at their...endogenous levels. HiBiT is compatible with both strong and weak promoters, and even modest endogenous ...quickly iterate, work across cell models, and even introduce mutations. This vector-based use of HiBiT provides...Proteins involved in endoplasmic reticulum quality control navigate a large and interconnected system of dynamic...the sensitivity of HiBiT with Addgene's quality control, support, and distribution capabilities. Give HiBiT...hepatocellular carcinoma in human tissue and in vitro. Oncology Reports, 50(5). https://doi.org/10.3892...

...A few years ago, researchers introduced what’s been called a major breakthrough in cloning technology...plant model systems. “The cloning is all done in vitro; it’s no different in mouse, human or plants,” explained...of an advanced molecular plant science lab.” Introducing GreenGate As Lohmann and his colleagues describe...describe in the PLoS ONE paper that introduced the kit in December, GreenGate includes the six most frequently... available through Addgene,” Lohmann said. “We strongly believe the free sharing of material is absolutely...