...ImageJ or similar software Reagents Purified recombinant antibody, 0.9–1.1 mg/mL SimplyBlue SafeStain,... bio-safe Coomassie G-250 stain (Addgene uses SimplyBlue SafeStain) and ImageJ software. The sample is...and reagents from other vendors should produce similar outcomes when using these protocols. However, please... Pour off the water in the sink. Add 20 mL of SimplyBlue SafeStain and incubate for 1 h with gentle agitation...agitation on a rocking platform. Pour off the SimplyBlue SafeStain in the sink. Add 100 mL of deionized...Analysis Use ImageJ (Link opens in a new window) or a similar photo software to determine the relative intensity...

...cloning sites, will result in similar sized bands, thus making this simple digest less informative. This...Plasmid Size -OR- Insert and Backbone Size The simplest form of diagnostic digest is one in which you ...products from each orientation are different sizes. Simply run the digest products on a gel and those clones...

...choice with minimal limitations. Background In its simplest form, PCR based cloning is about making a copy...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 18bases 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...

... (Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as TRI Reagent®, RNAzol®, QIAzol® ...how effective your cell lysis protocol is. While simple homogenization is effective for most mammalian ...Homogenize or lyse tissues or cells in TRIzol® or a similar product. For tissues: use 1 mL of TRIzol® per 100...

...bands needed to be separated - see FAQs below . Simply adjust the mass of agarose in a given volume to...How do you get better resolution of bands? A few simple ways to increase the resolution (crispness) of ...you get better separation of bands? If you have similarly sized bands that are running too close together...

...primers are mainly used for amplifying a small, simple fragment of DNA. On the other hand, a long primer...The structure of the primer should be relatively simple and contain no internal secondary structure to ...

...do you get better resolution of bands? A couple simple ways to increase the resolution (crispness) of ...you get better separation of bands? If you have similarly sized bands that are running too close together...

... Your Primers Primer design for LIC is often as simple as using the backbone manufacturer's suggested ...the first G on 3'-5' strand from cut site). For simplicity, only the 5' primer is shown here. Note: Use ...

...RNA extractions. Plus, centrifuges are useful for simply collecting liquid to the bottom of a tube. You ... can be about bench height. They often spin at similar speeds to their smaller cousins, but can hold much...

... al. , 2010). Number of fragments assembled simultaneously Multiple fragments can be assembled in one ...opens in a new window) Rabe BA, Cepko C. (2020). A Simple Enhancement for Gibson Isothermal Assembly. bioRxiv...

...can create thousands of identical copies using a simple set of reagents and a basic heating and cooling...Your 5’ and 3’ primers should be designed to have similar melting temperatures (Tm). Set the annealing temperature...

...and reagents from other vendors should produce similar outcomes when using these protocols. However, please...plate washer (optional) Scale Microsoft Excel or similar software Reagents Purified antigen Purified recombinant...

...Yuan, Y., Clark, P. M., Wang, Q., Martino, R. A., Sims, J. J., Bell, P., Raymond, A., Stanford, W. L., ... capsids cross the BBB via interactions with a similar protein called LY6C1. This protein is expressed...capsids were deposited by the Gradinaru lab. For simplicity, in each section we’ll list the Gradinaru capsids...Jang et al., 2023; Brown et al., 2021.) It has a similar transduction rate to AAV-PHP.eB in the brain, and...like the rhesus macaque, are approximately more similar to humans than New World Monkeys and rodents. Image...marmosets (Chuapoco et al., 2023). AAV.CAP-B10 shows similar behavior in marmosets as in mice, although the ... thymus, and transduce the heart and spleen at similar efficacy levels to AAV9 (Stanton et al., 2023)....

... to the BSL-1 requirements. Conclusion Although simple, following appropriate PPE protocol goes a long...

...tags or shRNAs to any vector (the procedure will simply differ in terms of primer design). Let's assume...

... trace file will give you more information than simply looking at the bases provided by your sequencing...

...pipette and avoid inconsistent pipetting with our simple tips Bacterial Transformations We walk you through...

...containers that can be autoclaved. Conclusion Although simple, following appropriate BSL-1 and BSL-2 protocol...

...sites . Sometimes enzymes cut sequences which are similar, but not identical, to their recognition sites....

...their own competent cells. This is a relatively simple procedure and is useful for performing low efficiency...

...the molarity of DNA ends available for ligation. Simply put, there are only two ends on any given piece...

...and reagents from other vendors should produce similar outcomes when using these protocols. However, please...

...and reagents from other vendors should produce similar outcomes when using these protocols. However, please...

...from academic viral vector cores. Our titers were similar to those reported by these institutions. This protocol...

... a stock solution of 100,000 ug/mL (100 mg/mL). Simply measure out 100 mg of ampicillin powder, add it...

...and reagents from other vendors should produce similar outcomes when using these protocols. However, please...

...and reagents from other vendors should produce similar outcomes when using these protocols. However, please...

...decide to do simultaneous IF instead (Figure 1B). To label all your targets in one step, you simply use a mixture... each target. Alternatively, you can perform simultaneous detection with a mixture of primary antibodies...appropriate secondary antibodies. The general process is similar to standard immunofluorescence, but with some extra...antibodies before labeling the next target. B) Simultaneous Multiplex IF, where all the primary antibodies... Brewer et al, 2022 for a sample protocol.)  Simultaneous IF Depending on your needs, your sample, and...consideration when deciding whether you can do simultaneous or sequential labeling is what conjugated secondary...with Alexa Fluor® 488) then you can’t use them simultaneously because you won’t know which label the signal...

...recapitulate in vivo conditions similar to humans. For example, due to their simplicity, yeast have become an important...often as every three weeks so scientists can simultaneously observe several generations of mice at a time... mice are more genetically and physiologically similar to humans than many other model organisms, they...study human diseases. Diseases in mice show many similar phenotypes to human diseases and have successfully...Yeast (Saccharomyces cerevisiae) Yeast, one of the simplest eukaryotic organism, is a commonly used model ...in breads and other baked goods! Yeast is cheap, simple and easy to work with as they can survive in a ...scientists to easily manipulate them. Scientists can simply inject one-celled embryos with DNA or RNA to edit...

...standardized constructs and tools for the rational and simple shuffling of DNA regions. Golden Gate Assembly ...Assembly Tool which is freely available online. Its simple interface can guide you through both experimental...with multiple point mutations NEBuilder HiFi can simplify multi-position site-directed mutagenesis by assembling...desired mutations. This same process can be used simultaneously for the generation of one or more mutations...With NEBuilder HiFi DNA assembly, it becomes as simple as mixing fragments together with linearized vector...laborious, requiring multiple cloning steps. You can simplify this process by using NEBuilder which requires...oligo. Generating an sgRNA construct becomes as simple as combining your favorite linearized plasmid with...

...confidence that they aren’t simply doing an experiment wrong. However, even simple procedures like pouring...Reproducibility Project: Cancer Biology have produced similarly low rates of reproducibility. Failed attempts ...someone else's new and incredibly useful technique simply by following the abbreviated methods section found...modified analyses on published data if you need to. Similarly, FigShare also makes it easier to share data through... Figure 1 where many scientists are performing similar experiments. Biology is messy enough that by chance...consolidate published and unpublished experiments on similar subjects in a single, searchable space. Finally... more likely that we’ll find a fluke when we’re simply trying to get our experiments to work, but part...

.... Thank you 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. Gutierrez...This post was contributed by guest blogger Derek Simon. The surgeries and standard molecular neuroscience...cemented to the animal’s skull using dental cement (a similar procedure as implanting a guide cannula). A fiber... which can control output of several lasers simultaneously, can be used to set the laser output without... Central PMCID: PMC3769938. 5. Iwai Y, et al. A simple head-mountable LED device for chronic stimulation...

... your lab bench, we need to (this is a little simplified): Find the appropriate glycerol stock within...that everything will go perfectly and our software simply follows a linear progression directing our techs... the most failure prone. In an academic lab, a similar situation could arise if a lab veteran who worked...failure: In our second example we’ve added some simple but critical logic. Some steps fail “hard”, they...the whole procedure. Other steps fail “soft” they simply need to be repeated. We also now have a good interface...contents from the container (of course this is a simple task in the lab, but the software should be mirroring... growth strains, vector backbone, etc.) and you simply don't have enough context to know what they really...

...provide you with some tips on how to automate some simple processes in your own lab. Automating bacterial...up a basic protocol on the Biomek FxP is pretty simple. By clicking a few buttons you can quickly tell...time. It follows all the same steps and uses the similar buffers as would be used for a mini prep, except...to automate these processes, but there are many simple steps you can take to automate processes in your...project in which you need to do a lot of aliquoting. Simply program the pipette with how much total sample ...Cap tubes with a micronic cap sealer. This is a simple table top device that has a roller to go over caps... of pushing them in one by one. It seems very simplistic and it isn’t electronic but it is one of the ...

...protein variants, Cas13, and Cas12a, to develop simple, portable, and inexpensive platforms to reliably...be detected on commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the...DNA or RNA of interest within a given sample is simply determined by the number of bands present on the...CRISPR Cas variant that can also cleave dsDNA similarly to Cas9 (Zetsche et al., 2015 ). Cas12a however...DETECTR. How does DETECTR work? DETECTR works similarly to SHERLOCK. Cas12a is targeted to a specific ...DETECTR could accurately distinguish between two similar types of HPV, HPV16 and HPV18, from human cells...Applications of DETECTR in diagnostics DETECTR allows for simple and efficient detection of nucleic acids in a mixed...

...coming weeks, but, in this post, we will outline a simple method for selecting the guide RNA, validating ...CRISPR Design tool supplied by the Zhang lab. This simple website invites you to input the target region ...happy with your selection, ordering the sgRNA is simple and easy. Several companies supply gRNA sequences...you gRNA in vitro first. In vitro screening is a simple and fast method for validating your selected gRNA...). Testing the efficacy of your gRNAs is then a simple matter of transfecting HEK293T cells with your ...the individual gRNAs in their PX330 plasmids (or similar plasmids for gRNA expression), one gRNA at a time...perfect primer sets to use to genotype your pups. A simple PCR with these primers will let you know if there...

...sometimes localize to unintended sites with sequence similarity to the on-target site. When a CRISPR-Cas system...targets by selecting a gRNA with low sequence similarity elsewhere in the genome. There are many predictive...Once you’ve selected a gRNA with low sequence similarity elsewhere in the genome to reduce off-targets...to consider your Cas enzyme. Even if there are similar sequences present in the genome, certain Cas enzymes...Candidate site sequencing Sites with high sequence similarity to the gRNA can be sequenced. These sites can...appropriate method for an activator or repressor Cas. Similarly, Cas ChIP-Seq would also identify all DNA binding...experiment due to their location (e.g. in a gene in a similar pathway, a growth factor, etc.) this control is...

...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...

...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...

...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...

...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...

...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 ...

...). 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...

...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...