...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....

...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...

...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...

...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...

...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...

...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...

...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 ...