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...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...

...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...

... 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  ...

...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...

... 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 ...

...Using a spectrophotometer is among the faster methods of quantification. Many spectrophotometers have an... you have multiple contaminating bands or a few strong ones, consider increasing the number of washes ... period of two weeks. After this time, quality control assays showed that the majority of antibodies are...references More resources on the Addgene blog Introduction to Antibodies Plasmid-based Recombinant Antibodies...Protein aggregation - Mechanisms, detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi.../j.cocis.2014.10.002. Randolph TW, Schiltz E, Sederstrom D, Steinmann D, Mozziconacci O, Schöneich C, ...

... 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...

...Optogenetics Guide γ-Retrovirus Guide Protocols Agarose Gel Electrophoresis Bacterial Transformation... direct shipments weren’t able to clear import control. Partnering with a distributor allowed us to share... How to Design Primers for PCR Agarose Gel Electrophoresis Inoculating Liquid Bacterial Cultures Restriction...Restriction Digest Analysis We also introduced guides — practical primers to help users get started ...we launched the Addgene YouTube channel to add a strong visual component to our educational resources. ... systems to allow for easier access to export-controlled items by streamlining permissions and paperwork...

...for this task. However, most lesioning methods destroy areas larger than intended and may unintentionally...to the neuronal networks involved in directly controlling muscle activity and coordinating movement, several...optogenetics to support these existing findings by controlling neurons in the mPFC with light. Activation of...inhibited by fear/anxiety. On the other hand, the control group preferred staying in the dark compartment...time the animal spends in the passive mode, the stronger is the depressive component of the animal behavior... such genetic study focuses on brain derived neurotrophic factor (BDNF). BDNF is a small protein involved...the same paper, the authors used lentivirus to introduce an shRNA to downregulate expression of miR124a...

...derived from the control trace file. On the plot, the green (edited sample) and orange (control sample) lines...cells, genomic DNA from both edited and unedited (control) populations is PCR-amplified and Sanger sequenced...determined by comparing the edited trace to the control trace. In the ICE algorithm, potential editing ...summary table. You can also use your browser’s Control+F or Command+F functionality to search for a particular...disagreement between the non-edited wild type (control) and the edited sample in the region around the...difference. Traces The Traces tab shows the edited and control, non-edited Sanger traces in the region around ...

...different scientific fields, and knowledge of how to troubleshoot problems at the bench. Specifically, roles in...loads of questions, and I also had to do some troubleshooting as advised by the technical support person....specificity of this antibody?” You will also help troubleshoot products, give general experimental guidance...the world easier. You should also demonstrate a strong willingness to help others with their problems,... initial contact, but is less suitable for troubleshooting or issuing replacement products. In order to...careers are open to experienced professionals with strong interest in the technical aspects of science and...work includes reviewing customer data sets, troubleshooting protocols, analyzing information, and solving...

... to introduce multiple genetic modifications in the same strain. MazF expression is under control of the... a GFP cassette, and introduced site-directed mutations, all without introducing unwanted selection markers...delivery vector can be integrated into the genome to introduce the genetic modification(s) contained within it...EcoRI and Bsp6I in Escherichia coli in 1995 when introduction of restriction-modification systems onto plasmids... able to monitor lacZ activity over 48 hours in vitro without antibiotic selection. In a subcutaneous ...

...unproductive conversations with internet trolls (urban dictionary for trolling: The art of deliberately, cleverly...to write our AMA submission to avoid rousing the trolls. 3. The AMA process was simple and well organized...while it can be anonymous, I can always block the trolls. It seemed like I would really have to open myself...moderators were on top of any comments that were at all trollish. Kudos to the moderators for keeping the quality...discourse. They even advised me on how to alter my intro to reduce the number of potentially hostile posters... the term “implicit bias” which is apparently a troll trigger phrase these days). Redditors ask serious...

...approach complete with freely available positive controls. Routinely test your cell line for mycoplasma ...freezing virus in a dry ice/ethanol bath or liquid nitrogen prior to storing. Know your target cells Labs ...These binding events can be impaired by the electrostatic repulsion of the negatively charged cell with..., A., Garnier, A. “Downstream processing of oncoretroviral and lentiviral gene therapy vectors.” Biotechnology...conditions on the production of lentiviral and retroviral vectors.” Human Gene Therapy Clinical Development...Cronin, J., Zhang, X.Y., Reiser, J. “Altering the tropism of lentiviral vectors through pseudotyping.” Current...Cleghorn G., West M.J., Wei MQ. “The significance of controlled conditions in lentiviral vector titration and...

... channelrhodopsin in the retrograde serotype, and a dTomato under the control of the E6 regulatory element...pAAV-CAG-emiRFP670 AAV1 Control Kiryl Piatkevich New viral tool pAAV-S5E6-dTom-nlsdTom AAV1 Control Jordane Dimidschstein...Wilson New serotype AAVrg = AAV retrograde (also called AAV2-retro) Looking for more viral vectors? ...

...CRISPR/Cas9 to cells for genome editing, and introducing inducible systems into cells, among many other...Desfarges & Ciuffi, 2012). One family of viruses, retroviridae, require integration for their life cycle to... during production (read our blog post on the retroviral life cycle for a more detailed description). ...concerning in clinical settings, as evidenced by retroviral gene therapy trials for X-linked severe combined...immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA...lessons from provirus and non-integrated DNA. Retrovirology, 1, 13. https://doi.org/10.1186/1742-4690-1-...resources on the Addgene blog Viral Vectors 101: The Retroviral Life Cycle Quick Guide to All Things Lentivirus...