... own RNA-dependent RNA polymerase into the infected cells. The EBOV polymerase replicates the viral genome...under control of a T7 RNA polymerase promoter sequence. Thus, T7 RNA polymerase also needs to be present...gene start) and a transcription termination/polyadenylation signal sequence (gene end). A minigenome is...replication and reporter gene transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome is capable...nucleoprotein, NP. NP associates with the viral polymerase, which is formed by a complex of two proteins...sense, single-stranded RNA minigenome. The viral polymerase complex then recognizes the encapsidated minigenome... order to replicate the minigenome, the viral polymerase initiates RNA synthesis at the 3’ end of the ...

...approach policy makers take in abusing climate science skepticism to promote harmful policies that benefit...move slowly, news about possible broad changes to policies seems to break everyday. Many researchers and ...participation in government, evidence to inform policies, a protected place to work, funding for science...medicines, technologies, defense, and governing policies are developed primarily by wishful thinking. The...rains, it was weather befitting of the ominous political climate in which American scientists find themselves...scientists found themselves not too long ago.   From policies that “muzzle” government scientists to the de-...Canada.  Outside the lab, she dabbles in science policy, public outreach, and restorative justice.  Follow...

...Sharing Policy Discussion PLOS’ New Data Policy: Public Access to Data PLOS’ New Data Policy: Part Two...advice about the scope of the policy. End of story? Not quite. The PLOS policy on data access is a formal...clarified what the policy intent was and provided an FAQ list to further explain the policy. The fact that...year when it announced its data access and sharing policy. Since early March, the open access publisher has...the research reported in PLOS publications. The policy was not an overnight revelation, rather it was ...reasonable. PLOS has since clarified their data sharing policy and recently announced that of the 16,000 manuscripts...agencies (e.g. Wellcome Trust, NIH Data Sharing Policies, etc.) and is founded on the principle that scientific...

...purification based on the polyhistidine tag. What is the polyhistidine tag? The polyhistidine tag is a string of...      Fig. 1: A sample containing a polyhistidine tagged protein of interest (purple) and several...to the Ni2+ and is immobilized along with a polyhistidine containing contaminating protein (orange). A...is successfully isolated.   Prior to the polyhistidine tag, alternative fusion tags were used, such...interfering with protein function. In contrast, the polyhistidine tag is very small and rarely affects protein...function (Bornhorst 2000).  One downside of polyhistidine tag-based purification, however, is the propensity... containing Co2+, have a lower affinity for polyhistidine than more commonly used substrates such as Ni2...

...conditions. Expression of an error prone polymerase I (PolI3M) is capable of mutagenesis in E. Coli, but... error prone polymerase to introduce mutations. However, by using nCas9 to direct PolI3m to a particular...EvolvR: (1) two additional mutations to PolI3M generated PolI5M which increased EvolvR’s mutagenesis rate... from bacteriophage T7 DNA polymerase increased the processivity of PolI3M, and thereby increasing the... system has two key components: the error prone PolI3M that’s fused to a nicking Cas9 (nCas9). Similar... site of interest. A gRNA is used to direct the PolI3M-nCas9 complex to a DNA site of interest, which ...which nCas9 nicks and then dissociates from. PolI3M then binds the nicked DNA, and extends it from the 3’ ...

...you need a high-fidelity polymerase. There are many high fidelty polymerases on the market; you need one...a DNA polymerase that produces blunt-ended PCR products (e.g. Phusion, Pfu, and Vent polymerases). Note... PCR followed by Restriction Fragment Length Polymorphism (RFLP) analysis (right). Introduction of restriction...amplified fairly easily by simply following the polymerase manufacturers’ PCR protocol. Be sure to adjust...plasmid template for site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired ... one with 5’->3’ polymerase activity (for amplification), 3’->5’ exonuclease activity (increases amplification...Note that certain polymerases such as Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary...

...      The RNA polymerase(s) RNA is transcribed from DNA using an RNA polymerase (RNAP). In bacteria...bacteriophage Constitutive, but requires SP6 RNA polymerase. SP6 polymerase has a high processivity. When used for...describes the combination of the promoter (RNA polymerase binding site) and operators (response elements...promoter region controls the binding of the RNA polymerase and transcription factors, therefore promoters...single enzyme; however, eukaryotes have muliple polymerases which are each responsible for a specific subset... used in expression systems for Drosophila. Polyhedrin General expression mRNA  Strong insect promoter... H1 small RNA expression shRNA From the human polymerase III RNA promoter Constitutive  May have slightly...

...path to science policy and science communication A career in science communication or policy requires broad... space of science education, communication, and policy. In this post, I hope to tell you a little more...technologies and as societal participants in the political process. My role at pgEd I have always been very...attending seminars in bioethics, public health, science policy, and science and technology studies (STS). I also...genetics research, their applications and regulatory policies; keeping tabs on the conversation about genetics...posts, lesson plans, or information briefs for policymakers. The rapidly changing landscape of genetics ...seminars, symposia, or courses in social, health or policy-related topics, as time permits. Also seek out ...

... bodies are a polymer of many copies of monomeric subunits. Unlike cytoskeletal polymers, however, R bodies...This post was contributed by guest blogger Jessica Polka, a Postdoctoral Research Fellow with Pamela Silver...force by dynamically rearranging into different polymerization states. Instead, R body monomers respond to...engineering objectives are possible in our recent paper (Polka and Silver 2016). First, we demonstrated that R ...mechanically with their environment.   Jessica Polka is a Postdoctoral Research Fellow with Pamela Silver...Harvard Medical School. Follow her on Twitter @jessicapolka.     References 1. Kanabrocki, J. A., Quackenbush...Protistology 48, 290–296 (2012). PubMed PMID: 22356923. 3. Polka, J. K. & Silver, P. A. A Tunable Protein Piston ...

...signaling, and transcription factor interactions (Kerppola, 2006). One of the first BiFC studies used the...construction, cellular expression, and quantification (Kerppola, 2006, 2013). These are described in more detail...RPACKIPNDLKQKVMNH have been used successfully (Kerppola, 2006). Flexible GS linkers may also work. You...analysis can be used in lieu of negative controls (Kerppola, 2006). Here, one of the endogenous PIPs is expressed...troubleshooting tips can be found in protocols by Kerppola (2006, 2013). BiFC vs. FRET: When to choose one...doi.org/10.7554/elife.38853 Hu C-D, Chinenov Y, Kerppola TK (2002) Visualization of Interactions among ... https://doi.org/10.1016/s1097-2765(02)00496-3 Kerppola TK (2006) Design and implementation of bimolecular...

... comes from using Taq polymerase for the amplification step since Taq polymerase leaves a single deoxyadenosine...mind that Taq polymerase has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading...and amplify your sequence of interest with Taq polymerase using your favorite PCR protocol.  2. Set Up ...to reduce error rates; however, proofreading polymerases will also remove all unpaired 3’ ends in your... 2. Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase....

...T4 DNA polymerase, Gibson assembly requires T5 exonuclease in combination with Phusion polymerase and DNA...LIC employed the exonuclease activity of T4 DNA polymerase to create longer, “chewed-back” overhangs of ...At that position, T4 would perform the favored polymerase reaction and subsequently stall due to the absence...contain the dNTP present in the reaction, or polymerization will occur at that position, preventing T4 ... plasmid are both partially digested using T4 polymerase in the absence of dNTPs. In the second step, ...advantage of SLIC over Gibson assembly is cost, as T4 polymerase is much less expensive than the enzymes required...

...to be hyperpolarized. The chloride pump NpHR brings in negative chloride ions which hyperpolarize the cell...positive or more negative.  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive...positive over a certain threshold, it becomes depolarized and will fire a signal. The non-specific cation...both cause ion movement that results in cell depolarization. They therefore function as a neuron “on switch...switch.” Neuronal inhibition: Hyperpolarization Conversely, when the inside of the neuron becomes more...

... protein mix by size Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the first...of a resolving gel composed of the crosslinked polymer acrylamide. In addition to the samples, a protein...membrane, usually composed of a nitrocellulose or polyvinylidene difluoride (PVDF) in a step called protein ...state while a western blot detects denatured polypeptide chains. The protein regions available for antibody...between a protein in its native state versus the polypeptide chain. Consequently, antibodies validated for...transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic...) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and...

...question you may have is whether a monoclonal or polyclonal antibody is better for your experiment. Monoclonal...antibody clone and therefore a single isotype while polyclonal antibodies contain a mix of different isotypes...tend towards more reproducible results. Since polyclonal antibodies contain a variety of clones and thus...secondary antibody depends on whether you are using poly- or monoclonal primary antibodies. Some secondary...secondary antibodies instead (B).   Since a polyclonal primary antibody contains a mix of different ...reactive secondary antibodies when you’re using polyclonal primary antibodies.  I hope this post has explained...

...Plasmid Multimerization Early studies on plasmid topology recorded plasmid multimerization in the naturally...and gene therapy development to examine plasmid topologies (e.g. supercoiled, open circular, linearized,...and can provide data on the quantity of various topologies and multimers present within a plasmid sample... Luo J, Guttman A, Thompson L. Vaccine Plasmid Topology Monitoring by Capillary Gel Electrophoresis. Curr...PMCID: PMC225270. Higgins NP, Vologodskii AV. Topological Behavior of Plasmid DNA. Microbiol Spectr. 2015...Michel ML, Schmeer M. Long-term stability study and topology analysis of plasmid DNA by capillary gel electrophoresis...

...downstream manipulations. Phi29 polymerase is a phage-derived polymerase that can perform whole genome ... approaches a host cell surface are complex polysaccharide structures, many of which are easily varied...fragmentation, repair sheared ends with T4 DNA polymerase (to create blunt ends) and ligate into a bluntly...manipulation, amplifying phage DNA with phi29 polymerase prior to fragmentation can greatly facilitate...into E. coli BL21 (DE3) cells (these have the T7 polymerase and a few proteases knocked out making them particularly...

...to express the transcription factors from a polycistronic cassette. However, it was unknown whether variations...variations in gene expression from these polycistronic cassettes could lead to comparable experimental...Woltjen Lab compared different variants of the polycistronic cassettes and discovered reprogramming discrepancies...variation in the Klf4 isoform (KLF4s or KLF4L). Polycistronic cassettes that contained KLF4s had overall diminished...reprogramming and global gene expression patterns. Polycistronic cassettes that contained the KLF4L isoform had...the observed reprogramming discrepancies. The polycistronic piggyBac transposon vectors tested in this work.... They are compatible with both agarose and polyacrylamide gels. They are shared through Addgene without...

...Plasmid tRNA Synthetase Expression Host pMAH-POLY Polyspecific aminoacyl-tRNA Synthetase Mammalian Cells ...protein of interest: you just don’t know it yet! The poly-histidine tag often used for protein purification...Green Tc: CCPGCC 50 40-72 Intact Cells Cy3NTAc Poly His  44 35-63 Plasma membrane protein, permeabilized...Lumio Red Tc: CCPGCC 48 38-70 Intact Cells Cy3NTAc Poly His  63 50-90 Plasma membrane protein, permeabilized...Lumio Red Tc: CCPGCC 54 42-78 Intact Cells Cy3NTAc Poly His  65 52-94 Plasma membrane protein, permeabilized...

...express fewer than four gRNAs, you’ll clone in a polyT-termination sequence for each unused promoter. This...transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being transcribed from...option described below, plasmids for making polycistronic gRNAs are also available from the Yang lab for...Comparison of Multiplex Strategies including Standard PolIII-gRNA cassette, Csy4-cleavable cassette, and PTG...Plasmids: These plasmids are similar to the Csy4 polycistronic system described above, except that they use...gRNAs are flanked by glycine tRNAs to create polycistronic glycine tRNA-gRNA (PTG) constructs. Eukaryotic...