...collaboration. The field already has comprehensive reference pages and curated databases for scientists including...discussion on the best strategies and protocols for different experiments.  In this blog post let's take a ... peer-reviewed and assigned a DOI that can be referenced. In addition the data published in microPublications...embryonic genes are individually depleted via RNA interference (RNAi). Scientists monitor the effects of RNAi...RNAi using four-dimensional contrast interference (DIC) microscopy and computer imaging processing. The... back frequently for what’s new and popular! References Hammarlund, Marc, et al. "The CeNGEN Project:... biology protocols Read our molecular biology reference ...

...remember: Keep Ca+2lm and Ca+2rry on! References and Resources References Nasu Y, Shen Y, Kramer L, Campbell...sensors are commonly used calcium reporters with different kinetic parameters. There are three GCaMP types...and Localization The calcium concentrations in different subcellular compartments can vary greatly. Additionally...measurements are more susceptible to artifacts and pH interference, and comparison between experiments can be more...sites per sensor (to decrease unnecessary calcium buffering and to increase sensitivity), and is a great FRET... also suitable for in vivo studies. There are different versions optimized for neuron-specific, astrocyte-specific...

...antibody for your application!  References and resources References Lu, Q. L., & Partridge, T. A. (1998... while polyclonal antibodies contain a mix of different isotypes and subclasses. They each have their ...of clones and thus the potential to bind many different sites on the target protein they tend to have ...polyclonal primary antibody contains a mix of different isotypes and subclasses, use a broadly reactive... be worth considering an antibody raised in a different species.      Table 1: Isotype considerations...targets, look for primary antibodies raised in different, and distantly related, species, and secondary...

...mammalian plasmid here and edit on! References and Resources References Kwart, D., Paquet, D., Teo, S. et...cutting efficiency — although both are of course preferred! When trying to strike that balance, you may wonder...sequence constraints, or at least has looser or different requirements. Luckily, there are some available...necessarily more flexible than NGG; they’re often just different (e.g. NGAG, NGAN, etc.). Additionally, alternative...enzymes certainly add more PAM options, none of them offer a one size fits all solution to the issue. Nor are...al). Additionally, SpG Cas9 with the same PAM preference of NG was isolated by the Kleinstiver Lab (read...

...in the body contain the same DNA but have very different structure and function, and can have a specialized...syndrome and Prader–Willi syndrome, which have very different disease phenotypes, are two fascinating examples... copies of chromosome 15 are inactivated, but different phenotypes result depending upon whether the improperly...development and progression of cancer through different mechanisms. New FRET based tools to study epigenetics...restriction enzyme digestion, DNA adenine methyltransferase identification, and bisulfite sequencing. ...the reporter consistently showed significantly different FRET levels between unmethylated and methylated... and treatment and improving patients lives. References 1. Ong-Abdullah, Meilina, et al. "Loss of Karma...

...numerous variants for the different types of opsins available and their differences and uses have been discussed... the different variants [1, 2, 4]. And, of course, the best way to learn about the many different opsin...about this in detail later). But what are the different steps in an optogenetics experiment and what are...available, instructions for obtaining a material transfer agreement, and list of vendors (such as the vector... core at the University of North Carolina) that offer his constructs. Importantly, control viruses (not... or follow him on Twitter @derekpsimonphd.    References 1. Tye KM, Deisseroth K. Optogenetic investigation...

...explore Myc’s role in cell proliferation and tumorigenesis.  Using two different synthetic peptides corresponding...antibodies, context is key.    References and Resources References Evan GI, Lewis GK, Ramsay G, Bishop... or the C-terminus, Evan, et al. isolated six different monoclonal antibodies (Evan, et al. 1985). Three... found that the N-terminal half of the antigen differs significantly between human and the other species...-9E10 variable regions and sticking them onto different scaffolds. But over the years, several alternative...

...variants—nuclease-competent or nuclease-null—in order to effect different perturbations. Consider, for instance, the challenge... both cutting and regulation of expression at different genetic loci within a single cell. One might imagine...use of “orthogonal” Cas9 proteins derived from different bacterial species, each of which interacts with... (1). Although this is possible, this strategy suffers from several limitations. Perhaps most importantly...expression (6). Altering the length of gRNAs targeting different sites of interest thus represents a straightforward...introduced together with truncated gRNAs targeting a different set of loci to induce simultaneous cutting and...DNA sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 ...

...library protocols you can use as a reference. Table 1: Examples of Different Pooled Libraries at Addgene.  ... plasmids in your library (Are the inserts of different sizes?), and the desired coverage for the library...For this reason, if your library has inserts of different sizes (such as a transcription factor overexpression...your growth time (e.g., 16-18 hours). Finding reference protocols There are many decisions to make when...pDNA Library Amplification protocol are useful references. Skim through our Pooled Library page to find...“one-size-fits-all” protocol! Each library is different, and you should always experimentally test the...

...you can make an informed choice! References and Resources References Bondeson, D.P., Mullin-Bernstein,... we will review what advantages degron tags can offer over other protein control methods and compare the...tools to manipulate protein expression. These tags offer several advantages over the alternative systems ... all the tags reviewed here, the SMASh tag does offer a few other unique features. If your protein is ... fluorescent Halo ligand can be added to media, buffer, etc., which will bind and light up a tagged protein...profile their effectiveness with over a dozen different proteins. In the end, you can’t predict with absolute...

...proteins that confer affinity for a target – and this serotype can be selected to preferentially target your...voltage indicators reviewed.   References and Resources   References Beck C, Zhang D, Gong Y. Enhanced... These types of sensors can have a variety of different components, but all contain a FRET pair, a genetic... genetically encoded component of the system to offer unique advantages depending on your experimental...powerful tool that swaps capsid proteins from different viruses to optimize them for targeting your cell...

...Forster Resonance Energy Transfer (FRET). Through the non-radiative transfer for energy between neighboring...requires careful selection of FP fragments and, preferably, some information about the structures of the...level, stability, and subcellular localization may differ from those of your BiFC construct, making it difficult...., do two proteins interact or not? — FRET is preferable when the temporal dynamics of PPIs are the focus...about science whenever he's not off the grid.   References Bischof J, Duffraisse M, Furger E, Ajuria L, ...plasmids and resources Visit our Molecular Biology Reference ...

...decorative" title="Check icon" %}   References and Resources References Hwang, J. M., Piccinini, T. E., Lammel...belongs to the penicillin family of antibiotics. It interferes with cell wall synthesis as its mode of action...beta-lactamase, just like amp is. Thus, the AmpR gene confers resistance to both carb and amp, due to their common...amp), and for some applications, the benefit it offers is marginal. Fortunately, amp can easily be replaced...phosphorylation of kan, which inactivates it. The NPTII gene confers resistance to several aminoglycosides, including...minutes). Pros: Widely available, cost-effective, confers mammalian resistance to G418. Con: Slow transformation...

...sample(s) in the same buffer if possible, or very similar buffers if not, since buffer components can affect...non-linear and you should use a different method of analysis. Different fields and assays have varying ...the assay is run, ideally with freshly prepared buffer. This is because standard curves vary depending...range covers a wider span of concentrations but suffers from decreased specificity, especially as it approaches...

...antibodies directly into your preferred buffer. If that’s the case, a buffer exchange can be performed using...downstream step, you’ll want to ensure that your buffer components are compatible with your conjugate and...you’ve collected your antibody in the appropriate buffer, it’s time to quantify it. There are several options...our Antibodies 101 blog series.  Resources and references More resources on the Addgene blog Introduction...Purification Protocol Addgene's Antibody Guide References Trimmer JS. Recombinant Antibodies in Basic Neuroscience...

...created with the same backbone but that contain different inserts. When you design primers to anneal outside...primer pair to screen for the presence of many different inserts. The downside: this type of primer does... The type of primers you use depends on your preferences. Either way, make sure to test your colony PCR...a standard PCR thermocycling program. One key difference is the plasmid DNA must be released from the ...and without an insert. These controls are quick references you can use when you run your PCR products out... an appropriate molecular weight standard for reference and to add a loading dye with glycerol to your...products have any mutations.  There are lots of different cloning strategies, but regardless of which is...

...PAM sequence requirements. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both...endonuclease domain, indicating that Cpf1 functions differently. Since Cpf1 loci are widely distributed across...could be adapted for genome editing. The use of a different nuclease could potentially overcome some of Cas9...pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning... cloning. Sticky-end mediated gene transfer would be particularly helpful for targeting non-dividing cells...shown the diversity of CRISPR-based systems. References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided...

...Labguru, Quartzy offers software designed with scientists workflow in mind and can offer a one-stop solution...create lists of your favorite protocols for future reference. The exchange currently boasts over 2,500 protocols... is a subscription-based software, Labguru does offer free trials and pricing options for academic labs... limited to within your lab group, Labguru does offer access to protcols from external vendors, such as...orders. Quartzy caters to life science labs and offers the service free of charge to scientists by allowing...101 articles on various plasmid features, and reference tables (i.e. antibiotic concentrations, commonly...

...preparative experiments.  We’re excited to begin offering this service to further contribute to our mission...and discovery.” Here are the details. Plasmids offered as cloning grade DNA As we begin testing out this...that this service will be helpful to the many different scientific communities using plasmid tools. Details... We will ship you  2 µg of DNA in 20 µl of Tris buffer suitable for molecular biology experiments such... our Plasmids 101 blog series Learn about the different grades of DNA  Read about the various ways to ...other beta projects Read our Molecular Biology Reference ...

...proteins also contribute to this regulation in different ways: methylating newly synthesized DNA, blocking...numbers can allow RepA to link iterons from two different plasmids, forming a pair of “handcuffs” that blocks...RepA proteins form complexes between iterons on different plasmids in a process called "handcuffing," which...Plasmids under stringent control of replication may differ in the specialized proteins or sequences necessary...was updated by Emily P. Bentley in June 2024.  References and resources Further Reading Quiñones-Valles...Replication. IntechOpen. https://doi.org/10.5772/19313 References Asano, K., & Mizobuchi, K. (1998). Copy number...molecular biology help with our Molecular Biology Reference Pages   ...