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...etiopathomechanisms of ischemic stroke, the most common type of stroke and the third leading cause of ...-Seeding Labs plasmid grant we are excited to introduce the 5 awardees and their research. These researchers... is necessary to improve tuberculosis care and control. In her current research, Sheen aims to identify...taraxacifolia (Willd), an annual herb of Western Tropical Africa typically known as wild lettuce. Aside ...of death and disability worldwide. An ischemic stroke occurs when arteries leading to the brain become ...of interesting combinations: biomedicine and astrobiology. During the last 11 years he has studied various...

...you can select an appropriate isotype control. An isotype control has the same constant heavy chain domain...heavy-chain constant region (see Antibodies 101: Introduction to Antibodies for a refresher). There are five...molecules are recruited by the antibody to help destroy and remove a pathogen. Different isotypes also ...doesn’t bind your antigen of interest. Isotype controls are used to make sure the staining you see is ... than nonspecific background staining. Isotype controls are used in antibody-based applications including...techniques that you can use antibodies for in our Intro to Antibodies post Check out our Antibodies 101:...

...The calcium is strong here….I can sense it….sound like a plot to an biology scifi movie? Or maybe it’...neurotransmitter, such as norepinephrine, or an electrolyte such as calcium. This varied component, which...For one, many viral platforms, such as AAV or retrovirus, can either permanently integrate and/or express...serotypes can be used to affect or determine viral tropism—the type of cells the viral particles infect. This... sensor, whether you choose lentivirus, gamma retrovirus, or AAV as your vehicle, we’ve got you covered...Resources References  Bhalla N., Jolly P., et al. Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60... Susanna Stroik...

...generation lentiviral packaging plasmid from the Didier Trono lab that expresses the HIV gag, pol, rev, and tet...pMD2.G. Find psPAX2. pMD2.G - Also from the Didier Trono lab is pMD2.G, a VSV-G envelope expressing plasmid...envelope protein for producing lentiviral and MuLV retroviral particles. It can be used with packaging plasmids...as pCMV-dR8.2 dvpr (lentiviral) or pUMVC (MuLV retroviral). Fun fact: published in 2003, this plasmid predates...generation lentiviral packaging plasmid from the Trono lab contains the rev gene. It’s meant to be used...generation lentiviral packaging plasmid from the Trono lab that contains the gag and pol genes. It also...shRNA. This scrambled shRNA is used as a negative control for mammalian RNAi experiments.  Find the scrambled...

...consequently the principal means by which CRISPR/Cas9-introduced breaks are repaired. The following factors are...ends tends to be the point where mutations are introduced. Repair of Cas9-induced breaks by NHEJ As illustrated...microhomologies”). NHEJ doesn’t obligatorily introduce indels. Given the end structure of the Cas9 DSB...hundreds of base pairs or more can simultaneously introduce a pair of chromosome breaks, and could result ...single strand break; when two such breaks are introduced near each other, in opposite strands. This “double...j.cell.2013.08.021 Waters CA, Strande NT, Pryor JM, Strom CN, Mieczkowski P, Burkhalter MD, Oh S, Qaqish BF...Video] Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens        ...

...that antibody works as expected, you will see a strong antibody signal in the retinal sample, but little...you see a similar antibody signal between your control and your knock-out sample, you should be concerned...antibody binding in the transfected cells but not in control cells. But (there is always a ‘but’), you should...encoding this protein, which we will call Negative Control Gene (NCG) (A). Then you lyse the cells and run... your antibody (B). Your results show a lovely, strong band from the cells transfected with your protein...well as a third unrelated antibody as a negative control (A). The two antibodies to your protein of interest...sample using the antibody and then uses mass spectrometry to identify the captured proteins. If the antibody...

...phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are critical reagents...circularization of dsDNA (6). Less commonly utilized in vitro, Taq DNA ligase will ligate only nicks (5-8). Taq...identifiable and quantifiable by capillary electrophoresis (CE) (see below). This method allowed screening...gene PCR-ligase detection reaction-capillary electrophoresis assay." Journal of clinical microbiology 45.6...PMC1933048. 16. Cheng, Changming, et al. "Electrochemiluminescent assay for detection of extremely rare mutations...mixed population of excess wild‐type DNA." Electrophoresis 34.9-10 (2013): 1415-1422. PubMed PMID: 23463388... PMID: 17466627. 25. Luo, Jianying, Donald E. Bergstrom, and Francis Barany. "Improving the fidelity of...

...anticipated outcomes? What are your controls? Will these controls enable you to interpret your results...e.g. an R01 from the NIH, in the United States) Strong letters of reference that attest to your talent...figures are missing? For example, are additional controls necessary? As you build your story, are additional...person makes time for you, as needed, this is a strong indicator that this person cares about your science... together, all of this means that you need to introduce yourself to and talk with more senior scientists...you and your research exposure. Use meetings to introduce yourself to and interact with the members of your...Medical Institute. His interests include the quality control of secretory proteins in the Endoplasmic Reticulum...

...identified seven genera in in vitro experiments, possibly because in vitro conditions did not optimally...situ Conjugation (MAGIC). With MAGIC, scientists introduce an Escherichia coli donor strain into the microbiome...antibiotic selection. The team tested this system in vitro and in vivo. They identified cells from 19 genera...from different bee species. BTK can be used to introduce fluorescent markers to visualize localization ...protein. The expression of this fusion is under control of the arabinose-inducible promoter. Cells are ...showed that this method can be used for both in vitro and in vivo studies. Find distributed cell division...

...eliminate fluctuations due to copy number by introducing genes of interest into the chromosome. But this...input signal that both positively and negatively controls the output. In this case, plasmid copy number ...gene increases its expression. However, if you introduce negative regulation by using a promoter that is.... placed a trans-acting RepA protein under the control of IPTG. Because RepA is a plasmid replication ...Segall-Shapiro et al. placed the genes under the control of a constitutive promoter and then moved them ...decreased. But when the genes were placed under the control of a stabilized promoter, deoxychromoviridans titers...the joys of a p-value < 0.05, having such fine control of gene expression is useful for many metabolic...

...autosomal dominant mutation in MYBPC3 that leads to hypertrophic cardiomyopathy. An adult male with a heterozygous...containing Cas9 and two different gRNAs, and electroporated them with an ssODN repair template in induced...wild-type copies of MYBPC3 (versus 47.4% of untreated controls.) 27.6% of embryos carried indels at the mutation...blastocyst stage at similar rates to untreated controls. Ma et al. performed sequencing of 23 potential... conducted whole genome sequencing in selected control and CRISPR-Cas9 injected embryos. In all cases,...challenging for homozygous mutations. Moreover, introducing new DNA seems impossible with current tools, ...applicable in many situations. In the case of hypertrophic cardiomyopathy and other genetic disorders, ...

...needed and when is it not? What are the proper controls needed to pressure-test your FRET assay to make...pyrrolysyl-tRNA synthetase Mammalian Cells pCMV-DnpK dinitrophenyl hapten Mammalian Cells pANAP AnapRS Mammalian...commercially available as Lumio Green and Lumio Red from Invitrogen (now ThermoFisher Scientific). These reagents...molecular distances (see Table). However, proper control experiments are needed to establish the dynamic...These experiments include producing a positive control construct where the donor and the acceptor fluorophore... each other on the same protein and a negative control experiment conducted on donor-only labeled protein...biarsenical− tetracysteine motif determined by NMR spectroscopy." Journal of the American Chemical Society 131.13...

...All three can introduce multiple base pair deletions, but CRISPR/Cas9 can also introduce point mutations...homologous recombination. A targeting vector is electroporated into embryonic stem cells and this will insert...joining, the newly inserted gene may have errors introduced to the nucleotide sequence during the DNA repair... in more detail in part 2 of this blog series) controls the cell types in which the conditional recombination...engineered mouse model allows gene expression to be controlled through a drug or small molecule that regulates..., Cre is fused to the ligand binding domain of estrogen receptor alpha (Esr1). Mice are fed or injected...

... A fast blue-light inducible Cre for precise control of bacterial gene expression By Meghan Rego Recombinases...recombinases to manipulate genomes, scientists can control expression of their genes of interest. While recombinases...their activity can be fine-tuned and precisely controlled through the use of optogenetics. Optogenetic ...authors demonstrate that CRISPR-Casɸ is active in vitro as well as in human and plant cells. Casɸ...assay for mammalian cells. It's based on the introduction & resolution of two DSBs in a fluorescent reporter...serotype AAV5.  Get the channelrhodopsin here Astrocyte specific tight affinity glutamate sensor from ...from the Sanford Boye lab with enhanced retinal tropism. Find the upcoming serotypes here  ...

... affinity terminator may be desired for multi-cistronic constructs where high termination efficiency is...structure. It is thought that poly(A)s are used to control the cellular concentration of regulatory RNAs and... and may additionally act as a quality control mechanism to rid the cell of mis-folded RNAs. Eukaryotic...virus 40 late polyadenylation signal by using in vitro analyses. Molecular and Cellular Biology. 1992. ...Ther. 2008. PMID: 18627247 Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck Hepatitis Virus Posttranscriptional...Enhances Expression of Transgenes  Delivered by Retroviral Vectors. Journal of Virology. 1999. PubMed PMID...Resources on the Addgene Blog Learn how the Promoter Controls Gene Expression Learn about New Cloning Techniques...

...stimulation itself, the output of the laser must be controlled. As discussed above, each opsin variant has slightly...the A.M.P.I Master-9 Pulse Stimulator, which can control output of several lasers simultaneously, can be.... Pulse generation (TTL pulse) is a standard electronics protocol not specific to lasers or optogenetics...powerful alternatives to the Master-9 that only control one or two lasers are also available. Optimal stimulation...loss of laser power. More recently, wirelessly-controlled optogenetic implants have been reported by several...References 1. Gutierrez DV, et al. Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate... Kim TI, et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics...

...do not let the panelists introduce themselves. I do a 1-2 minute introduction. If you chose good panelists...and will play a different role so her ability to control the event is what is important. Choose 4 panelists...logistics To make a really lively discussion, I strongly suggest asking the panelists not to use any slides...it (lapel mics are best). The moderator takes control It is the moderator's job to prevent anyone from...talking, so they will easily take 5 minutes to introduce themselves and, voila, 1/2 of your time is gone...publicity. Good panels do not waste time on long introductions. The moderator asks questions, calling on 1-...

... of plasmids, they are generally electroporated into electrocompetent cells to ensure the highest possible...chemical transformations is less than that of electroporation, but an advantage is that chemical transformations...protocol, the size of the cuvettes your lab’s electroporator uses, etc. The DNA amount can be anywhere from...DNA in 100 μL of cells over four individual electroporations. The specifics of this can be based on the...5,000,000 30 bp At least 200 ng Liquid Electroporation Protocol (DOCX, 15.8 KB) Barcode SPLINTR... CRISPR Preparing Pooled Libraries NGS Quality Control for Pooled Libraries Resources on Addgene.org Addgene's...

...SARS-COV-1 plasmids. Export control license process    An export control license, once approved, allows...repository, including rabies plasmids, are export-controlled materials in the USA, where Addgene is located...applicable research project when purchasing export-controlled materials. Addgene will then apply for the license...   Due to recent regulation changes, an export control license for rabies materials may be required for...application process.  More information about export control licenses is available through the US Bureau of ...

... they then confirmed by PCR. Subsequent mass spectroscopy studies allowed for the identification of proteins...Fujii Lab generated a transgenic mouse line and retroviral expression system for using this approach in ...analyzed, the Fujii lab also developed another "in vitro" form. In this form, the CRISPR complex, consisting...biotinylated gRNA, were successfully used in this "in vitro" form. Any tag system can be used to isolate genomic...expressed can cause facioscapulohumeral muscular dystrophy (FSHD), a disease for which there is currently... isolation of DNA fragments and chromatin by in vitro enCh IP technology using recombinant CRISPR ribonucleoproteins...as an RNA-guided platform for sequence-specific control of gene expression." Cell 152.5 (2013): 1173-1183...