...In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time...cloned into a plasmid of interest. You can use similar processes to add overhangs to your insert of interest...primer design and the PCR process itself are very similar to those outlined in our restriction cloning post...TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but we need to use the reverse complement to get... get PCR amplification. We can start similarly, taking the final 18 bases of the ORF, including the stop...primer. If you are amplifying from a plasmid or simple template, there is very little chance for mis-priming...

...genome editing.  For a few reasons, Cpf1 is a simplified system for editing multiple targets compared ...polycistronic transcript  Requires co-transfection of Cas9 Nissim et alTsai et al PTG Cassettes transfection gRNAs...s ability to process its own pre-crRNA arrays simplifies the crRNA cloning process. For cloning, Zetsche...oligos that consist of direct repeats and crRNA.  Similar to a jigsaw puzzle, the oligos were designed with...latest developments in CRISPR technologies.  It’s a simple and effective method with multiple applications... vivo, transfection, viral transduction) for simultaneous multi-gene editing with a single crRNA array...

...lesser-known Cas enzymes while also highlighting their similarities and differences to Cas9. DNA targeting Cas...self-process pre-crRNA into crRNA). The size and simplicity of Cas12a makes it easier to package for therapeutic... even a single mismatch in the target sequence Similar to Cas12, it is also significantly smaller in size... size than traditional Cas9. Another similarity to Cas12a is that Cas14 also indiscriminately cleaves ...opposed to permanent edits to the genome. Cas13, similar to Cas14, also indiscriminately cleaves ssRNA when...pre-crRNA self-processing. Functionally, it is very similar to Cas13 and has been proposed to be useful for...

...through sequence homology. Similarly, Golden Gate assembly allows one to simultaneously complete an ordered ...described by Chaokun Li and colleagues. Here you simply PCR-amplify your vector and your insert, DpnI digest... way to generate a wide variety of constructs. Similar, Gateway cloning (discussed below) can run on the...relatively new to the cloning game, however, or simply looking for a more streamlined approach, you might...1. Li, Chaokun, et al. "FastCloning: a highly simplified, purification-free, sequence-and ligation-independent...

...antibodies may have similar emission spectra, meaning they will emit fluorescent light at similar wavelengths ...fluorescence for both at similar levels, meaning that your populations will ‘look’ similar on your flow plot....between the two of them because they emit at a similar wavelength. However, if your flow cytometer reads...is if the names of the fluorescent markers are similar, you will need to compensate between them (although...

...some simple solutions available for researchers to deal with all of these problems. The simplest and best...forming dimers in cells. Why should you care? Three simple ways a dimeric FP could ruin your day (and experiment...cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might think EGFP is unlikely... a cell. Unfortunately, things are not quite so simple. Concentration is the number of molecules within...form heterodimers (i.e. EGFP and mCherry). These simple strategies can help you make better fluorescent...

...columns had similar DNA yields. Additionally, columns regenerated up to five times had similar DNA yields...plasmid miniprep protocol from Addgene follows a similar workflow as a column-based plasmid extraction kit... This method provides DNA yield and quality similar to that obtained with commercial kits, but in less...cellulose-based method was faster, cheaper, and had a similar level of sensitivity. Figure 2: Steps to ...waste footprint. The Zhao and Deng labs developed a simple method for regenerating silica columns that come...

...). Fluorescent tags may also be used, but for simplicity we will discuss the more common enzyme-based ...Direct ELISAs A direct ELISA is the quickest and simplest of all varieties. In this method, users immobilize...source of cross-reactivity.   While the method is simple and saves time, it also has several drawbacks. ...   Indirect ELISAs The indirect ELISA process is similar to that of a direct ELISA, but the indirect method... drawbacks of the competitive ELISA method are similar to those of the direct method. Namely, since the...Method Advantages Disadvantages Direct Fast and simple protocolReduced chance of cross-reactivity Higher...

...tool is in such a small package. But don’t let the simple presentation deceive you; pooled libraries can ...titer with a colony formation titering assay or similar protocol. After functional titer is determined,...our more popular libraries, you might receive a similarly-sized aliquot from an amplification done by Addgene...first step will be to amplify the library. This is similar to amplifying a plasmid: transform E. coli with...place to start is with a protocol for a library of similar size and backbone.  Verification Once your library...representation of your amplified library should be very similar to that of the original library. Like amplification...

...linear epitope derived from the P and V proteins of Simian virus 5 (SV5) (Randall et al., 1987). The epitope...solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using tag-specific monoclonal...and characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences...differences between human, canine and simian isolates. J Gen Virol., 68 (Pt 11), 2769–2780. https://doi.org...target multiple genes in M. abscessus either simultaneously or stepwise, achieving mutation efficiencies...first use of CRISPR/Cas9 in M. abscessus. The simplicity and efficiency of this dual-plasmid approach ...

...composer. View his blog drsimonsaysscience.org or follow him on Twitter @derekpsimonphd.    References 1. Stuber...This post was contributed by guest blogger Derek Simon. The actual experiments you do will be determined... also models reinforcement but is considerably simpler than ICSS. The test chamber is divided into two...but before you do any type of behavior analysis, simply take a few animals and stimulate them for 30min...that pun worn out yet?) your research. Derek P. Simon received his PhD in Cellular and Molecular Biology...

...portion of the plasmid containing the mutation). Similar results can be obtained with PCR screening using...large as ~6 kb can be amplified fairly easily by simply following the polymerase manufacturers’ PCR protocol...into a larger band (not shown). As a footnote; a similar approach can be used to identify Cas9/CRISPR-induced...will evade detection in the initial RFLP analysis simply because the introduced restriction site is also...site is located close to your target site, you can simply amplify and mutate this smaller portion of the ...want to make your protein non-functional, you may simply introduce a frame-shift mutation. This can be achieved...

...binary systems above if they all behave very similarly?” The simple answer is that sometimes it is necessary...transcriptional activation domain VP16 taken from the herpes simplex virus. tTA binds to promoters containing TetO elements...cumate-inducible gene expression system, which functions in a similar manner to the Tet system. The transactivator configuration...interrogate the activity of uncharacterized promoters. Similarly, placing UAS upstream of a transgene permits directed...dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev...

...Densmore’s lab at Boston University have greatly simplified the process of generating multiple E. coli gene...GMAP - A new assembly platform for generating both simple and complex DNA constructs Have you ever wished...Sanger sequencing, respectively. The speed and simplicity of GMAP allows scientists to easily design, generate... tend to show their limits when it comes to simultaneously modifying multiple loci (multiplexing). Currently...(MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand DNA (ssDNA...in a broad range of strains. Thus, pORTMAGE simultaneously allows genome editing and mutant library generation...genome editing in multiple organisms thanks to its simple, easily modifiable RNA-guided targeting mechanism...

...They are classic parametric tests and one of the simplest statistical tests available. Tailed t-tests If...groups (two samples and a control, for example) simultaneously, with up to two independent variables. This...that matter. However, this is oftentimes an oversimplification. If your independent variables are affecting... you only look at 2 or 3 pairwise comparisons. Similar to family-wise error, is the false discovery rate...positives/negatives, but they all generally yield a similar result and are appropriate to use if all sample...with Ordinal Dependent Variables: A Monte Carlo Simulation Study. Front. Appl. Math. Stat. Volume 2 (2016...

... by a similar function. After analysis, CRISPR classifications were placed into a fairly simple hierarchy...classification system relies on a combination of sequence similarity, phylogenetic analysis, neighborhood analysis... the same class and type don’t share sequence similarity but are grouped due to mechanisms of action and... ensure every unit of the complex expresses at similar levels. It was only recently that researchers began...subdivided into subtypes A–G. These subtypes have similar compositions and mechanisms, with the biggest differences...

...antibodies are less likely to cross-react with other, similar proteins due to recognizing a single epitope of...monoclonal antibodies here. Recombinant antibodies Similar to monoclonal antibodies, recombinant antibodies...internal control for Western blots, shows that a similar concentration of cell lysate was added to each ...   Enzyme-linked immunosorbent assay (ELISA) Similarly, ELISA is used to detect the presence of a single...expression of multiple proteins on the same cell simultaneously as well as compare between samples. Learn more...localization within the cell or tissue as well. Similar to flow cytometry, you’ll use antibodies to stain...

... Many thanks to our guest blogger Derek Simon! Derek P. Simon received his PhD in Cellular and Molecular...composer. View his blog drsimonsaysscience.org or follow him on Twitter @derekpsimonphd.    References 1. Tye...This post was contributed by guest blogger Derek Simon. Optogenetics is spreading through the neuroscience...generation. Archaerhodopsin (Arch) is activated at a similar spectrum as HR and also results in membrane hyperpolarization...

... abundance of thousands of protein variants simultaneously  With increasing sequencing power, scientists...the effects of sequence variants on abundance simultaneously called VAMP-seq, for Variant Abundance by Massively...the a bacterial periplasmic binding protein, one similar to the protein which gave rise to the iGluSnFR ...artificial leaf replica means creating a “leaf” with similar permeability of water and nutrients and colonization... to a second population. Last, Bernach will simultaneously monitor cell cycle and growth rate using TIMER...

...endless ways and everyone finds their own. Here, I simply outline what works for me, and I hope that it is...will keep you from repeating your work or making similar mistakes in the future. Stay organized Set up...experiments can be found in each folder. This can be a simple .txt document. Record the name, date, and location...track of your exact changes. Alternatively, you can simply make a note in your methods section about the edit...result was, so try not to do this. These are the simple rules that I try to follow in my own research. ...