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...did not result in significant recombination. If you do see recombination, we recommend ensuring the cells...pooled libraries can lead to issues such as recombination and loss of plasmid diversity, so Addgene strives...strives to distribute aliquots of the original library whenever possible. In this blog, we’ll discuss ... lentiviral backbone might be more prone to recombination, while a library with a large backbone might...diversity of the library is maintained and reduce recombination during the amplification process. In liquid ...factor overexpression library) or is prone to recombination, we recommend amplification on solid media. ...may not have amplification protocols attached.  Final step: validation  Remember, there is no “one-size-fits-all...

...researchers test many different combinations of promoters, terminators, solubility and purification tags...connectors, cDNAs, and more to find the perfect combination. The EcoFlex Kit has a wide array of DNA parts...EcoFlex a great option for those interested in recombinant protein purification and metabolic engineering...expression, the system includes a toolkit to express recombinant proteins within the mitochondrial matrix.   ...specialized system with parts including site-specific recombinases, regulatory elements, developmental regulators... examine the effects of different promoters, terminators, enhancers, and cross-cassette interactions in...localization and transport studies in HEK cells, recombinant protein purification in bacterial cells, and ...

...validation data, or follow the references to find the original publication and ensure that it includes validation...-lot. The highest reproducibility comes from recombinant antibodies (rAbs), which are produced from plasmids...plasmid sequencing. While rAbs are monoclonal, recombinant multiclonal antibodies — mixtures of different...re-purchasing your primary antibody with the new reporter. Finally, each new conjugate will have to be tested to ensure...It is recommended for small to medium proteins. Finally, there are dry/electroblot transfer systems available...range, they cannot be read with equal accuracy. Finally, they degrade quickly and need to be read fairly...compatible with stripping and probing.   Imaging At the final step of your western, you’ll have two choices: x-ray...

...Cas13a: An RNA cleaving CRISPR nuclease Cas13a was originally identified in 2015 (Shmakov et al., 2015). They...enzymatically “active” state rather than reverting to an inactive state like Cas9 or Cpf1. As a result, Cas13a will...sequence of interest (red) is amplified using Recombinase Polymerase Amplification (RPA) or Reverse-transcription...crRNA targeting the sequence of interest and an inactivated fluorescent RNA reporter. If the target sequence... whether the target sequence is present in the original pool of nucleotides.   What are the potential...regulating expression of specifically targeted RNAs. Finally (and perhaps most interesting), Cas13a’s propensity...-targets. They subsequently used catalytically inactive dLwaCas13a as a programmable RNA-binding protein...

...presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more stable and brighter version...essentially mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its protein...possibilities. For instance, further selection of Spinach with a target metabolite creates an aptamer that...but it still had limitations in vivo. Using a combination of SELEX and fluorescence-activated cell sorting...also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium dependence...Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey, Samie R. “Fluorescent RNA Aptamers ...Activity with GFP Resources at Addgene.org Find the Spinach Plasmids at Addgene Find Broccoli Plasmids at Addgene...

...hinders experiments is mycoplasma contamination. Mycoplasma contamination has been shown to arise from a ...Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention... growth and behavior. Signs of a mycoplasma contamination can be subtle and may include reduced saturation..., labs should routinely test for mycoplasma contamination. There are several commercially available mycoplasma...PCR-based approaches to detect even low levels of contamination. For those labs that prefer a more economical....C., Drexler, H.G. “Detection of mycoplasma contaminations.” Methods in Molecular Biology 946 (2013). ...

...coli strains are not usually well suited for recombinant protein expression. Many challenges can arise...coli. We will review the potential pitfalls of recombinant protein expression and some of the most popular...or are enzymatically active. For this reason, recombinant proteins are typically expressed in E. coli engineered..., which reduces proteolysis of the expressed recombinant proteins. lon protease: Strains where this is...proteins. hsdSB (rB- mB-): These strains have an inactivated native restriction/methylation system. This means...plasmids utilize inducible promoters, which are 'inactive' until an inducer such as IPTG is added to the...to make protein.  The DE3 lysogen/T7 promoter combination is the most popular induction system. The DE3...

...dietary supplements Dr. Mrinalini Nair, University of Baroda, India Dr. Mrinalini Nair, Associate Professor... viruses. Now focusing on plant biology, she coordinates the Network for the Genetic Improvement of Cowpea... order to manage insect pests and to support sustainable agriculture. For this project the expression ...LIC cloning vector and the pOKD plasmids. The recombinant Cry proteins will then be screened for toxicity...oxidase. The goal is to determine potential water contaminants and to subsequently develop technologies to ...who applied, and to Seeding Labs who helped us coordinate and promote the awards. References 1. Martinez-Klimova...

...AAV5 Recombinases Stanley Thomas Carmichael New viral prep pAAV-EF1a-fDIO-Cre AAV5 Recombinases Esteban.... These preps drive expression of either Cre recombinase or a fluorescent marker in the various cell types...serotype pAAV-hSyn-Cre-P2A-dTomato AAV PHP.eB Recombinases Rylan Larsen New serotype AiP13778 - pAAV-...pAAV-AiE0450h-minBG-iCre(R297T)-BGHpA (Alias: CN3778) AAV PHP.eB Recombinases Jonathan Ting New viral prep AiP13779 - pAAV-AiE0779m...minBG-iCre(R297T)-BGHpA (Alias: CN3779) AAV PHP.eB Recombinases Jonathan Ting New viral prep AiP13781 - pAAV-AiE0452h-minBG-iCre...pAAV-AiE0452h-minBG-iCre(R297T)-BGHpA (Alias: CN3781) AAV PHP.eB Recombinases Jonathan Ting New viral prep pAAV-CAG-GFP ...

...produce recombinant proteins at high levels. In a case where clinical applications of recombinant proteins...wondered where human embryonic kidney (HEK) cells originated from? Or why they are such a commonly used cell...difficult to determine its true tissue origin. Originally, it was assumed HEKs were a common cell type ... necessary, HEKs are human in origin, so they eliminate non-human PTMs which is a concern with other protein...cultured human cell lines are susceptible to contamination by mycoplasma and bacteria, but HEKs are also...., HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors. Front Bioeng Biotechnol...

... mountain – with many routes up and only one destination – a career in the sciences is more like an intergalactic...a simple egg is packed with information. I am fascinated with how developmental programs unfold, absorbing...comparative anatomy showed me that some of the most fascinating biology is found in weird and rare organisms ...stick to vertebrates alone.  And a longstanding inclination to be a teacher was looking for an outlet. I ...Association for Women in Science); others were visiting seminar speakers. My eye was caught by a couple of unusual...how punctuation can be wildly funny as well as financially critical. Those who work on the publishing side...the right audiences and how open access affects financial models in information publication. Take a look...

...technology. In each section below, we cite the original paper but link to plasmids from the Promega Plasmid...similar constructs available at Addgene. Read the original paper for more detail on whether the authors incorporated... attempts to purify or overexpress proteins inappropriate in discerning their true roles. Work conducted...receptor (EGFR), a transmembrane receptor tyrosine kinase essential for cell proliferation signaling. CRISPR...angiogenesis through its principal receptor tyrosine kinase VEGFR2 (also known as KDR), orchestrating blood-vessel...cb3002478 Kamada, Y., Tateishi, H., Nakayamada, U., Hinata, D., Iwasaki, A., Zhu, J., Fukuda, R., & Okiyoneda...10.1111/bph.15426 Promega Corporation (2025). Illuminating Transmembrane Proteins Using HiBiT CRISPR Cell...

...conductor receptor (CFTR) by homologous recombination in cultured intestinal stem cells of patients with cystic...This post was originally written in 2014 by Kendall Morgan and updated in 2022 by Lucie Wilson. Lucie...CRISPR/Cas9 could be used to edit several autosomal-dominant mutations that have been connected to the early...Chinese Academy of Sciences found that mice with a dominant mutation in a gene that causes cataracts could... chose to explore its potential in mice with a dominant cataract disorder caused by a single copy of a... "Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients...

...pAAV-hSyn-DIO-hM3D(Gq)-mCherry Bryan Roth   Recombinases Recombinase systems such as Cre-lox allow for more...types, recombinases allow you to further restrict gene expression to areas in which the recombinase and ...and construct are coexpressed. Three new recombinases from the Cepko Lab are now available as AAV1. CRE-...constructs 69570 and 69571 together encode the N and C terminal segments respectively of a GFP dependent Cre. ...to form a functional Cre enzyme that induces recombination of lox sites. This system takes advantage of...

...efficiency.  Finding recombinant psychrophilic enzymes for the host The expression of recombinant proteins for...conditions decrease the potential for microbial contamination. Additionally, the high activity of these enzymes...well under suboptimal conditions. This means recombinant proteins can aggregate or precipitate out as ...would allow the stable folding and expression of recombinant psychrophilic proteins. As the figure below shows... as engineering and regulating psychrophilic recombinant proteins.  To infinity, and beyond! Keeping ...brainstorm with each other. The event also featured a seminar on “Tools to increase scientific reproducibility...

...interest: The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate integration...line that stably expresses Gag-Pol and/or Env, eliminating the need to deliver these genes in trans by specific...feature may be useful for the transduction of terminally differentiated (non-dividing) cells. Both lentiviruses...produce additional virus. Are γ-retroviruses self-inactivating (SIN)? They can be. Plasmids that encode SIN... LTR Subcomponents: -U3 -R -U5 LTR; Long terminal repeats; U3-R-U5 regions found on either side ...U5 and the rest of the provirus. 3’ LTR Terminates transcription started by 5' LTR by the addition...written and updated by multiple Addgenies. It was originally published in 2018, in the Viral Vectors 101 eBook...

...With no blue light, the channelrhodopsin remains inactive and neurons don't fire. With blue light, ions ...minimally-invasive, Frances Arnold’s lab and Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins... To overcome these challenges, the Arnold and Gradinaru Labs developed a machine-learning model to help...information about parental channelrhodopsins to create recombinant channelrhodopsin sequences. The model first removed...their success in vivo. Therefore, the Arnold and Gradinaru Labs expressed ChRgers in mice to see if they ...broad transduction of the nervous system which eliminates the need for intracranial injections. When packaged...Bedbrook CN, Yang KK, Robinson JE, Mackey ED, Gradinaru V, Arnold FH (2019) Machine learning-guided channelrhodopsin...

...second day of Antibody, Addgene gave to me…  A recombinant anti-GFP!    🎶On the third day of Antibody, ...fourth day of Antibody, Addgene gave to me…  A recombinant Anti-Arl13b!    🎶On the fifth day of Antibody...sixth day of Antibody, Addgene gave to me… A recombinant Anti-PSD-95!    🎶On the seventh day of Antibody...eighth day of Antibody, Addgene gave to me…  A recombinant Anti-DYKDDDDK!   🎶On the ninth day of Antibody...tenth day of Antibody, Addgene gave to me…  A recombinant Anti-APP!   🎶On the eleventh day of Antibody... of Antibody, Addgene gave to me…  A growing recombinant antibody collection!     ...

...Miyawaki, Griesbeck, Heim, & Tsien, 1999), monitoring kinase activity (Ni, Titov, & Zhang, 2006), quantifying...tRNA suppression competes with natural chain termination at stop codons, expression levels of these tagged...acceptors, then biarsenicals are a good option. Originally developed by Roger Tsien and co-workers (Griffin...compounds such as British anti-Lewisite (which was originally developed to treat heavy metal intoxication in...insert your protein of interest, resulting in a C-terminal fusion of a tetracysteine tag. In addition, since...so don’t be afraid to try novel, unpublished combinations of labeling strategies. FRET experimental design...Roger Y. Tsien. "Specific covalent labeling of recombinant protein molecules inside live cells." Science281.5374...

...vectors and 5 lentiviral Destination vectors. These vectors can induce combinatorial constitutive or inducible...vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is a technology that can ...can be used to induce site-specific recombination events and is widely used in the field of mouse transgenics...(Plasmid #44246) Expression of a catalytically inactive (dead), human codon-optimized Cas9 under the control...deposited by Dr. Tyler Jacks. It carries CRE recombinase and a puromycin resistance cassette. No time... higher titer of lentiviral particles than the original lentiCRISPR v1. Special note from the Zhang lab...