...and medium can all affect copy number. Some ori's are temperature sensitive; others ori's can be "tricked...result, stretches of DNA that are rich in A-T pairs can be separated more readily at lower temperatures and...balance between positive and negative regulation and can be manipulated with mutations in the replicon. For... Senior Scientist Marcy Patrick says researchers can ask themselves a few questions to get started: Will...mind that plasmids with low to medium copy numbers can still express massive amounts of protein given the...product. The E. coli strain Most E. coli strains can be used to propagate plasmids, but endA- E. coli ...

... library containing lentiviral vectors the iBARs can be detected by next generation sequencing.  In order...different lengths, the density of cells that are labeled can be altered to dense (45% of cells labeled), intermediate...sparse (4%) levels. SPARC is a versatile system that can be used in many cell types and organisms including...allow scientists to measure calcineurin dynamics and can serve as a drug-screening platform to identify new...fusions with tTALE to “stain” DNA. TALE proteins can bind specific DNA sequences which brings the attached...comparison to intercalating fluorescent dyes which can cause photo-induced DNA breakage and structural deformation...deformation or photo-induced damage. These fusions can also function in high salt conditions - up to 100...

...to ensure high-quality library distribution. You can also use these tips as a starting point when you ...composition of the final recovered DNA. This bias can be counteracted by growing the cells on solid plates...for the Brunello library is shown in Figure 2. You can use a small PCR product because the plasmids in the...the same output: a spreadsheet of read counts. You can use these counts to confirm the representation of...plasmids that were amplified preferentially). You can also rank the plasmids by abundance, scaled to 1,... calculating the area under the curve (AUC), one can gain a sense of the uniformity of the library distribution...verifying pooled plasmid libraries. Pooled libraries can be a powerful research tool, but their proper use...

...fluorescent proteins that can be distinguished from one another and that can be imaged with the optics...there are many fluorescent protein combinations that can be imaged together, but this also means that the ...determination of whether two fluorescent proteins can be separated from each other requires knowledge of... nm / em 474 nm) and YFP (ex 514 nm / em 527 nm) can be imaged together but CFP and GFP (ex 488 nm / em...there are techniques, like spectral unmixing, which can be used to separate the fluorescent proteins, but...particularly not the most recently published ones), they can be a good starting point. In many cases it is sufficient...

... Cas9 can be reprogrammed to serve as a regulator of gene expression. Its catalytic domains can be mutated...genetic edits. Cas9 is a DNA binding protein that can be directed to virtually any genetic locus when complexed...break (DSB) stimulates DNA repair mechanisms that can be exploited to either inactivate a gene or introduce...this nuclease-null or “dead” Cas9 (dCas9) variant can then be fused to effector domains such as transcriptional...activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation of an RNA hairpin...4). Taken together, these minor gRNA alterations can be exploited to quickly and inexpensively generate...benefit of the use of truncated guides is that they can be delivered into systems that already express nuclease-competent...

...detection can be done by the naked eye. This makes cloning quite a bit easier since you can see if your...non-specific binding to proteins can inhibit their activity. ShadowR can be used as a FRET acceptor in combination... 1). Figure 1: The sea anemone's red color can be used for mimicry. Image: Bernard Spragg. Advantages...chromoprotein, after transforming them into your cells, you can simply look for blue colonies to determine which ...other substrates for screening. Thus chromoproteins can be used as markers in cases where cost and resources...chromoproteins used in the lab come from eukaryotes, they can be difficult to directly use in bacteria, where most...’ve learned what chromoproteins are and how they can be used, why not add some color into your research...

...Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can infect dividing and non-dividing...meaning they can be used to create virus that is capable of infecting target cells, but cannot produce any...Are retroviruses and lentiviruses the same thing? Can I package lentivirus with retroviral packaging plasmids...dividing and non-diving cells while retroviruses can only infect dividing cells. When choosing the appropriate...target cell or tissue needs to be considered. Q2: Can I use lentiviral transfer vectors for transient transfections...2nd vs. 3rd Generation Lentiviral Plasmids Q3: How can you tell 2nd- and 3rd-generation transfer vectors...requires Tat to function. Third-generation plasmids can be packaged with either system. Please note that ...

... therefore GFP cannot be downsized by deleting residues. Only about 10 amino acids can be deleted from...proteins that bind endogenous fluorescent co-factors can be used. Examples include the LOV domains that bind... GFP is sensitive to acid The chromophore of GFP can exist in a protonated and a deprotonated state (Tsien...proteins emit light. The pKa of fluorescent proteins can be accurately determined in vitro by measuring the...Chatterjee et al., 2013; Hilaire et al., 2017). GFP can only be used for tagging proteins GFP fusion proteins... interest with an additional protein module that can report on location. This approach enables the study....g. DNA, RNA, and lipids. Still, these molecules can be indirectly visualized by using protein domains...

...of error that can impact your western blot, and depending too much on normalization can amplify other ... precisely evenly across the entire blot. So how can you accurately compare different lanes? In relative...loading control. Assumption #2: The loading control can be accurately quantified In western blotting, the...qualitative interpretation of this gel is misleading. We can see why on the S-shaped curve. On the protein abundance...susceptible to error, since tiny changes in intensity can reflect massive differences in protein abundance....assumptions for different loading controls So how can you address these errors without making incorrect...loading control. Assumption #2: The loading control can be accurately quantified Most housekeeping proteins...

...our materials are available to any country that we can export to — so far, we’ve shipped materials from ...transit time for each courier we contracted with so we can always offer the fastest shipping. However, there...broadly helped increase access in the country, and we can see a growth in material distribution after we partnered... popular Chinese social media platforms.  As you can imagine, shipping to so many countries means that...distributors who have expertise in a specific country can quickly reduce a large number of barriers. Though... from visitors in 218 countries. In Table 1, you can see our most popular educational resources for each...videos, where showing exactly how to execute a step can provide needed clarity (Table 1). Our most popular...

... What I can promise is that my suggestions can help you make the best case for your candidacy. A major...what you think you can accomplish. No matter how many times I say this, candidates regularly try to convince...hesitate to email me at [email protected]." You can find his original blog post below! --Ed.  Eight ... your friend in a lab outside of your department can understand your cover letter and research statement...the experts ask you about the details and then you can display your deep knowledge of your experiments and...question is whether there is chalk. Sometimes. It can also be a slide deck, markers, etc. Chalk or markers... succinct. Slide decks leads to the opposite and can suffocate an exciting idea in a swamp of unnecessary...

...The edit in the chromosome can now take place. After editing, pTargetF can be cured from the cell by inducing...another sgRNA targeting another site of interest can be used to make other edits. Once you have all the...plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et al., 2021...is superior in several ways: The pEcCas plasmid can be operated at 37 ℃ for faster growth of its E. coli...required and the BsaI linearized pEcgRNA backbone can be prepared in large amounts and frozen for future...nt oligos to generate new pEcgRNA. pEcCas/pEcgRNA can be applied not only in coli K-12 strains, but also...alternative versus plasmid-based expression which can be unstable and lack precise control over copy number...

...violet light can penetrate. How can I try using photoswitchable kinases in my experiments? You can find the...of interest, but there can be a lot of static since other signaling networks can also be non-specifically...intermediate messengers. Optical control of kinase activity can provide greater spatiotemporal resolution than pharmacological...ERK KTR isn’t phosphorylated and red fluorescence can be found in the nucleus. For testing kinase inhibitors... of signaling vs the delayed effects because you can precisely control the timing of kinase activity. ...

...packaged in a non-enveloped icosahedral capsid, that can be used to express genes of interest in cell and ...non-pathogenic, do not integrate into the host genome, and can be specifically targeted to a number of different...rAAV. For those interested in heftier genes, you can coinfect with multiple viruses to express genes or...interested in expressing multiple small genes, you can design single rAAV vectors with multiple genes packaged...tissues is one of its biggest advantages. Specificity can be achieved using specialized promoters, Flp or Cre...combination of promoters, gene switches, and/or serotype can also be used to ensure specificity; this is particularly...second strand of DNA before expression of their cargo can begin, which all takes time. Though the time delay...

...are a few things you can do to make the experience less stressful. First, you can make arrangements with...since one story usually has more than one theme and can be used to answer multiple types of questions. Writing...search for “questions to ask an interviewer,” you can find lists that have lots of general questions about...interviewer by looking at their LinkedIn profiles. This can help spark a more natural conversation while also...companies have relaxed dress codes for interviews. You can also ask HR for clarification on the dress code for...manager and others, if you have their emails. You can use notes you took during your interviews to help... talk for a remote interview. Fielding questions can be hard during this talk since you’ll be sharing ...

...Oftentimes, it can be quite difficult to ascertain what your audience knows, but the following tips can help:  ...'re in the midst of sharing your science - which can drive away even the most enthusiastic of learners...load?  Everything! Here are some common areas that can use up your audience’s mental bandwidth.  Jargon ... you at the most basic of levels.  Concepts This can be obvious, in that a lot of science communication...communicate your key points well enough that the audience can take that knowledge and continue building on it as...audience has is a major factor in how quickly they can understand and process your information. A class ...learn to observe and adjust on the fly.  Once you can think about everything you’re asking your learners...

... Yet little explanation is given for how someone can or should demonstrate this quality.   I am often ...high quality. Their work involves coordination and can require technical mastery. Scientists need to be ...hire and fire to make sure the team is the best it can be. Managers must ensure that their teams are working...operating in a different role?  I think the secret sauce can be summed up in one word – initiative.  Initiative... listed a few of my favorites. Additionally, you can find more suggested resources on this topic and get...Reads: On Leadership (Articles on Leadership, also can be purchased individually) ...

...although sequences with G/C content outside this range can still be optimized (Anzalone et al., 2019). Test ... pegRNAs whose 3′ extension begins with a C base can cause unwanted base pairing (left), while pegRNAs... What to edit The sequence you choose to install can influence the success of your prime edit. If possible...synthesized strand before heteroduplex resolution, which can lead to indels (Anzalone et al., 2019). Figure...left), but “bubbles” of 3 or more mismatched bases can often evade MMR (right). Created with BioRender.com...PE3/PE5 approach. Figure 7: Nicking sgRNAs can be designed to target edited DNA, so that nicking...scaffold sequence (orange) and the genomic target can increase the odds of incorrect editing outcomes, ...

...overhangs, stat? GetSet can do that for you, complete with predicted fidelity. You can specify number of overhangs...highest-fidelity overhang set it can find for those parameters. For even more specificity, you can exclude specific sequences...overhangs with a high potential for mismatches, so you can redesign individual pieces as needed to improve fidelity...rather than pre-designed sets, so identical searches can return multiple answers. You might want to take advantage...fusion sites to split it into multiple fragments. You can define the number of fragments, and the “search windows...constricted to a specific number of fragments, you can also ask it to give the maximum or minimum number...52 fragments may be near the upper limit of what can be accomplished with Golden Gate Assembly—even when...

...region or nucleus. Just optimizing AAV expression can take months (or longer), yet the process is often...detailed in the primary literature, and as a result can leave scientists under-informed with respect to how...cell types used for packaging viral particles (6), can influence vector performance. Open science initiatives...especially for AAV data, that may not be the case. We can all acknowledge that negative data is something every...getting this information out into the public where it can be utilized by others. Addgene’s AAV Data Hub is ...evaluate how small changes in vector administration can influence AAV expression properties.  By providing...generally captured in the literature, scientists can use the Data Hub to compare different methods and...