...with a 6x His tag. Requires a strain with T7 RNA polymerase. Amp resistant. More expression vectors for protein... of the pB1H2w derivatives as a fusion to RNA polymerase subunit ω. Transcription factor binding to a ...

... 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....

...The key is T4 DNA polymerase, which has 3′→5′ exonuclease activity. T4’s polymerase activity can be thwarted...thwarted if no dNTPs are available to polymerize, allowing the slower exonuclease activity to proceed.... of the chosen nucleotide, T4 will switch to polymerizing and then stall due to the absence of the other...dNTP present in the exonuclease reaction or polymerization will occur at that position, preventing T4 ...target vector. 5′ overhangs can be generated by T4 polymerase (left branch); by annealing two separate PCR ...advantage of SLIC over Gibson assembly is cost, as T4 polymerase is much less expensive than the enzymes required...

...Michael Rosbash 128046 pGL3basic luciferase and renilla_polyA Firefly/Renilla Insertion of 5' promoter/enhancer...luciferase James Wilson 101156 T7-CMVtrans-FFLuc-polyA Firefly T7 Expression of firefly luciferase from...

...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...

...mRFP-Clc Clathrin-coated vesicles Clathrin, light polypeptide mRFP Ari Helenius 70217 pEGFP-GFP11-Clathrin ...Clathrin-LCa-EYFP Clathrin-coated vesicles Clathrin, light polypeptide EYFP Xiaowei Zhuang 14437 mRFP-Rab5 Early endosomes...

...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...

...lot number while you’re at it! Polyclonal or monoclonal Both polyclonal and monoclonal antibodies can ...can work well for a western blot. Polyclonal antibodies, which bind to multiple sites on an antigen, are...protein — can be used to reproduce the benefits of polyclonal antibodies. Because these mixtures are defined...are not as commonly available as monoclonal or polyclonal antibodies, but it’s worth a look to see if they...Precast gel Increased cost Save money Polyclonal antibodies Higher non-specific binding; lot-to-lot...available; cost varies widely Increase signal Polyclonal antibodies Higher non-specific binding; lot-to-lot...

...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...

...the past several years, it seems that the more polarized (negative) the action potential of a cell, the...differentiated and quiescent it is, and the more depolarized (positive) the cell is, the more embryonic, stem...cation channel) light-activated channels to hyperpolarize tumor cells, which resulted in tumor regression...activation, it stays open for a longer duration. Hyperpolarizing chloride channel SwiChr++ - A channelrhodopsin...and gap junction protein-mediated cues control polarity during planarian regeneration." Developmental ...

...negatively-charged membranes (Davis et al., 2002). Polybrene is one such cationic reagent that is commonly ...efficiency by 10-fold (Davis et al., 2002). However, polybrene is highly sensitive to freeze-thaw cycles and ...be stored in single-use aliquots. Never use a polybrene solution that has been thawed more than once. ...in cells that are sensitive to the toxicity of polybrene (e.g., hematopoietic or primary cells), and has...., Jeffrey R. Morgan, and Martin L. Yarmush. "Polybrene increases retrovirus gene transfer efficiency ...

... impossible to interpret. Polishing the fiber optics with diamond polishing paper of sequentially increasing... second key point; the fiber optics need to be polished to significant smoothness to prevent drastic loss...its output with a light meter before and after polishing. Efficiency of the implant should be at least ... output. The same considerations apply to the polishing and preparation of the patch cable (although you...

...sufficient to seed polymer formation as fusion protein concentration increases. The polymerized fusion proteins...dimerizing FP has potential to form large stable polymers of dimers. 3) FRET biosensors that contain two...dimerization coupled with POI dimerization can lead to polymerization of the fusion protein. A dimeric fusion protein...resulting in false positives [2, 4]. Solutions to the polymer problem There are some simple solutions available...

...not suited to generate polycistronic containing multiple identical polyadenylation signals or promoters ...fluorescent proteins, promoters, enhancers, polyadenylation signals, inducible gene-expression sequences...promoter (to drive high-level expression) and a polyA signal (to terminate transcription) and the four...constructs were then combined to create a single polycistronic 15 kb insert. The final construct was then introduced...

... how much they get paid. Institutional salary policies are one indicatator of how salaries may changes... 2014, 89% of institutions had a minimum salary policy: of those, 51% of institutions set their postdoctoral...National Postdoctoral Association Institutional Policy Report 2014). However, ~30% set their minima lower...lower. Furthermore, 7% did not enforce these policies. It is unclear how many postdocs are at each of ...only is this in direct contradiction to explicit policies set up other institutions (like University of ...

...target protein, so they are referred to as polyclonal. Polyclonal antibodies have the advantage of more robustly...find more details about polyclonal antibodies on our Antibodies 101: Polyclonal Antibodies blog post. Monoclonal...advantages and disadvantages compared to each other. Polyclonal antibodies These antibodies are derived from ...

... boxes Some vendors take back their expanded polystyrene (a.k.a. Styrofoam or cooler boxes). We get these...receiving temperature-controlled reagents. Expanded polystyrene will likely end up in a landfill, but at least...with CO2 emissions. NEB takes back expanded polystyrene boxes for free (in the U.S.). Their cooler boxes... 1976! Millipore Sigma takes back expanded polystyrene boxes for free (in the U.S.). After you receive...

... excessive PCR cycles or low fidelity Taq DNA polymerase, the construction of specific sequences for base...believed that the exonuclease activity of the polymerase generates sticky ends in the PCR reactions. These...any subsequent steps. Using high fidelity DNA polymerases, I have never had any mutations introduced into...efficiency is highest when using high fidelity DNA polymerases like phusion. An 18-cycle PCR leads to the most...

... a duplicated SV40 polyadenylation region instead of the synthetic polyadenylation signal present in pGL3...system for light-activated CRISPR-Cas9 effector, Polstein and Gersbach fused the light-inducible protein... accessible for many potential applications. Polstein LR & Gersbach CA, Nat Chem Biol 2015 Mar;11(3)...recruits up to 24 copies of a protein to a target polypeptide. Multimerization in the SunTag system occurs ... simplifies the process of creating complex polycistronic lentiviruses. The MuLE toolbox contains a series...constitutive promoters, 2 includible promoters, 3 polyA terminators and various pieces for inducible expression...UAAs via a re-assigned stop codon (TAG). This polyspecificity enables the site-specific introduction of many...

...just upstream of the final ITR.  polyA  The polyadenylation or polyA signal aids in the nuclear export...translation, and promotes RNA transcript longevity. The polyA is located at the end of the AAV genome, just upstream...

...use a high fidelity polymerase to minimize mutations. The fidelity of the polymerase becomes more important... million bp depending on the polymerase used. No matter which polymerase you use, it is important that...

... be shared across other proteins. Conversely, polyclonal antibodies - which are a heterogenous mixture...antibodies, their cost is higher than that of polyclonal antibodies. For example, it takes longer to develop...than it does to immunize an animal and recover polyclonal antibodies (Lipman et al., 2005). This is reflected...Trudel LJ, Weis-Garcia F (2005) Monoclonal Versus Polyclonal Antibodies: Distinguishing Characteristics, Applications...

...cloning capacity of ~8 kb, so either a single polycistronic vector or multiple single-gene vectors can be...retroviral vectors. This makes it possible to use a polycistronic cassette that expresses multiple reprogramming...factors can be delivered either as a single polycistronic transgene or with four different adenoviruses... ~14.5kb minicircle expressing a 2A peptide polycistronic cassette comprised of OCT4, SOX2, LIN28, NANOG...help mediate their transduction, such as the polyarginine peptide described in Zhou et al and Kim et ...

... Traditionally, they have been made as either polyclonal antibodies or monoclonal hybridomas, but those...those techniques have several drawbacks. Polyclonal antibodies produced in animals may be variable among...& Plückthun, 2015a).   Figure 1: (Top) Polyclonal antibodies are produced in animals and consist...Recombinant antibodies offer numerous advantages over polyclonal antibodies and traditional hybridomas.  First...

...biomolecules to the gel and add gel components to polymerize. Step 3: digest the sample to allow for even ...components penetrate the sample, how well the gel polymerizes, and the level of digestion. All of these factors...of epitopes during sample anchoring and gel polymerization - issues that can be minimized through tweaks...tweaks to these steps. But even under ideal polymerization and digestion conditions, loss of signal often...