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...simple presentation deceive you; pooled libraries can be quite challenging to use! In this post, I’ll walk...  When you buy a pooled library from Addgene, you can either order a DNA pooled library, which contains...lab, there are custom viral prep services that can prepare the libraries for you.  Starting from plasmids...verification, and lentiviral production steps before you can start transducing your virus. Let’s start there! ...functional titer is determined, larger lentiviral preps can be used for screening-scale transduction of cells...mutated by the viral pooled library. These cells can then be used for genetic screening, followed by sequencing... don’t. The size and composition of your library can drastically impact what conditions it needs to properly...

...customer problems. Technical support can be highly rewarding but can also be a good jumping off point for...obvious at rather late stages (i.e. postdoc level) and can include the pressure to publish in high-impact journals...there that require exactly what life science PhDs can offer: vast practical lab expertise, experience in...may have to answer technical enquiries such as: “Can I use this product under conditions different from...might be involved in such a role. In addition, it can be advantageous to speak multiple languages - that... For particularly tricky cases, team discussions can often lead to otherwise unobtainable solutions. To...essential for success. For possibly this same reason, it can be one of the more constant and safe corners in a...

...tight-knit team like that can and will be a little gut-wrenching, but you can get that feeling of camaraderie...I'm looking to see if there are any resources that can help with the transition.” The lab-to-desk-job culture...have as well. Making the transition from the bench can be jarring for the unprepared, and unfortunately,...academic research lab involves a few key features that can be difficult to replicate in other work settings:...experimental dry spells. Losing that sense of discovery can be hard since it’s frequently why many people get...most enjoyable aspect of their chosen career. You can confidently call yourself a Scientist. You work in...getting used to. Just remember: change is good. You can experience a real onboarding process. Unless you ...

... homologous regions can be encoded in the primer sequences. Multiple plasmids can be included in a single...epitope tag can be moved to the N-terminus of a protein-coding sequence, or a mutation can be introduced...However, while not widely known, plasmid assembly can be achieved in vivo using a bacterial recombination...reagents or purification kits, it is low cost and can be immediately adopted by any laboratory. All procedures... Fragment generation - Any linear DNA fragments can be assembled in vivo as long as they have homologous...Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid modification, from...whole vector. The new codon and homologous region can be included at the 5’ end of either primer. Subcloning...

...network is to be always thinking “How can I help?” and not “What can you do for me?” One of the easiest ... connections can connect you to people at that organization. This type of connection can help you gather...interviews.  Pro tip: You can learn a lot from the company LinkedIn page. This can include news, inclusion...messaging, not everyone likes to. A direct email can be the best way to get in touch if you have business...t really know, follow instead of connecting. You can see what they post and occasionally comment on their... people, this option is in the “More…” menu. You can also follow companies and organizations to see their...fair game for connecting to others.  Pro tip: You can change the option for sending LinkedIn messages to...

...packaging vectors for larger genes - they can hold up to 8 kb, while AAV can only carry around 4.5 kb. Different...Behaviors you can study using viral vectors By modulating neuronal gene expression and activity, you can use viral...development of viral vectors, behavioral neuroscientists can now precisely control where and when certain genes...required, because seemingly tiny external factors can have a quite noticeable influence on the outcome ... several days preceding the experiment. Handling can however negatively interfere with certain tests and...and affect behavior. A fact to consider: Rodents can hear ultrasonic frequencies (> 20 kHz, produced from... left behind from a preceding animal (ex: urine) can be distracting. If it is not cleaned, animals tested...

...measure how many of the viral particles produced can actually infect cells. These assays typically involve...several commercially available p24 ELISA kits that can quantify p24 levels in a matter of hours. A major...quantification tend to be overestimates since they can include free p24 and defective viral particles. Direct...backbones; once validated these universal primers can be used to titer any lentiviral prep that shares ...p24 assay, titering via measurement of viral RNA can overestimate the amount of infectious virus in a ...defective particles. Studies have found that values can be 10-1000 fold higher than for functional assays...from amplification of the original plasmid which can over-inflate results. A packing vector deficient ...

... transductions can offer benefits such as tissue-specificity, high efficiency, and can be selected to ...which can’t be easily achieved with alternative methods. Ready, set, transduce! Making virus can sound... or AAV. Then, your desired DNA to be integrated can be cloned into the vector that you have selected....resulting in functional virions. These viral particles can infect other cells, but since the majority of the...production cell line (depending on the virus), virus can be collected for the transduction step. Viral particles...viral collection fairly easy. Producer cell media can be collected, centrifuged to remove any producer ...rogue HEK-293 cell). This virus-containing media can then be placed on whatever cells you would like to...

...fluorophores, and can bind and light up epitope tagged proteins in vivo. These probes however can be expensive...optimized elements of an antibody probe, scientists can now more easily visualize single molecules and newly...protein sequences called epitopes and scientists can use these to tag their proteins of interest. Antibody...experiments demonstrated that the HA frankenbody can be used to amplify fluorescent signals in in vivo...effective in sensitive cells such as living neurons, and can be fused to other fluorescent proteins and tags including... to get started with your experiments! What else can you do with the HA frankenbody? Another large advantage...minutes in living cells, meaning that scientists can track the movement and localization of single HA-...

...technique can be used as a preliminary validation on your bulk population. This way you can ensure you... you can determine if it is worthwhile to isolate individual clones from that population. You can additionally...such as CRISPResso can help with the data analysis. If NGS is not an option, you can still detect off-target...systems generate a deletion large enough that it can be visualized by running your PCR on a gel. It’s ...sure your PCR amplicon isn’t too large so that you can easily visualize the size shift you anticipate. A... a new recognition sequence. In these cases, you can screen using PCR and a restriction enzyme digest....endogenous restriction enzyme sites? Never fear! You can introduce a silent, passenger edit to serve as a ...

... of a codon. You can domesticate the sites yourself using PCR, but gene synthesis can save you time. Using...about the Modular Cloning (MoClo) system and what it can do for you, and you’ve chosen one of the MoClo kits...together in the same tube at the same time, however, it can prove itself to be quite the mental puzzle if you... the pipetting! MoClo nomenclature and structure can be quite confusing for new users, and your cloning...and easy-to-understand guided manner. These videos can be especially useful when designing new primers to... simulate the restriction–ligation reaction. You can input the sequences of the parts and acceptor vectors...your sequence to improve assembly efficiency. This can be done through domestication, which is the modification...

...Twitter because I can get a little opinionated and, while it can be anonymous, I can always block the ... (short for “Ask Me Anything”) on r/Science. You can see some of Joanne’s comments on the AMA process ...Reddit is a volunteer-organized platform where users can participate in forum discussions about a wide variety...scientific fields. They also host AMA forums where users can ask designated scientists anything about a particular...active community of people interested in science. You can see some of the conversations going on within the...place for us. We’re definitely glad we did - you can see some of the stats from the AMA to the right -...The AMA process was simple and well organized. You can check out the r/Science AMA Submission Guidelines...

...problem, how can one little change make a difference? Individual changes to our behavior can lead to cultural...where I’ll list some easy but effective habits you can incorporate to reduce your lab consumption and increase...posts. Making small changes in your daily habits can feel ineffective or unimportant. With so many big...cultural shifts that can have bigger impacts. For example, if you joined an office where everyone used ...cups. By initiating little changes in ourselves, we can both influence others to change their behavior and... the lab Here are a few easy and fast things you can start doing right away to reduce your consumption...makes a big difference. Clean out freezers so you can make room and don’t have to keep buying more freezers...

...proteins is that the tag can alter the structure of the protein. In some cases this can lead to changes or ...staining can be beneficial when expression levels are low as multiple secondary antibodies can bind to ...protocols? If this sounds like you, then flow cytometry can help you overcome these challenges and make these...When combined with cell sorters, this technology can be used to isolate a specific subset of cells in ...fusions In addition to visible light, flow cytometers can have a variety of fluorescent light sources. When...cytometers however are highly versatile and some can accommodate up to 30 different colors. This lends... In addition, the level and timing of expression can also be affected by the tag.  Protein expression,...

...because organic solvents can be quite harsh - lipids and soluble proteins can be lost during fixation ...the protein, which can disrupt protein structure and thus structural epitopes, but can reveal linear epitopes...organic solvents can be used to simultaneously fix and permeabilize a sample. But you can also use organic... you might come across a couple other terms that can cause some confusion. Paraformaldehyde: Many IF ...Along with preserving your sample, cross-linking can block the epitope of some targets, preventing your... to be washed away. Furthermore, these chemicals can also alter the structure of proteins, which could... improve antibody binding for some targets, they can destroy it for others. That said, organic solvents...

... Overexpression of fluorescently tagged proteins can lead to improper protein localization, protein aggregation...fluorescent protein fusion. CRISPR genome editing can help accomplish this goal. The power of this system... specified by a guide RNA (gRNA) sequence. Users can design gRNAs to induce the break at a specific genomic...pathway, a new user-defined DNA sequence (like GFP) can be inserted at the DSB. CRISPR has allowed scientists... contain your newly tagged gene. Most insertions can only be detected by labor-intensive processes like...high-throughput targeting vectors. These vectors can be used to tag endogenous genes and simultaneously...connector and tag donor plasmids from the library can be used to generate functionally unique repair templates...

... Alex Schier’s Lab. gRNAs can be in vitro transcribed from ssDNA, which can be generated by commercial...incorporated into the genome     Off-target effects can occur due to prolonged Cas9 expression     The requirement...compared to plasmid-based expression of Cas9/gRNA and can be used for most of the current genome engineering...using RNPs for CRISPR experiments. The RNP method can often be used in cells that are difficult to transfect...transfect, such as primary cells. Using RNPs can also alleviate difficulties with protein expression that...protein and a gRNA from a commercial vendor, this can often be expensive. An alternative approach is to...experiments using RNPs is the variety of methods that can be used to deliver a Cas9-gRNA RNP. This advantage...

...example) and can be analyzed using Addgene’s Analyze Sequence tool. Excellent, how can I check if the... of web pages the plasmid page is nested in. You can see in the example above that the bread crumbs trail...plasmids with similar names in the collection, but you can use this name to find this plasmid from our search...out this video for a walkthrough of the ways you can search our website). A quicker way to find this plasmid...out the plasmid's structural features and see if I can easily clone my gene into it? Your best bet is ...plasmid maps as part of the deposit process and these can be found in this location as well. These maps make...provide plasmid maps whenever possible. GenBank files can also be uploaded onto our plasmid pages, but these...

...these biosensors can be found at Addgene. Split FPs – some FPs (e.g. GFP, Venus) can be split into two...Photoactivatable / Photoconvertible: These proteins can switch their color when activated by a specific excitation...wavelength. This means that the emission wavelength can change. In a few cases, the initial state of the ...proteins change their color over time. Therefore, these can be used as “timers” for cellular processes following...they will form the full FP and fluoresce. Split FPs can be used to determine the proximity of two proteins.../em peak. Therefore, choose FPs that your system can excite, and detect the emission. For example, if ...yellow light). Note that some combinations of FPs can cause an effect called FRET (fluorescence [or Förster...

...backbones and specific primers you can use to confirm your clones. You can also find a list of popular sequencing...gRNA insert or not. A blue-white screen, however, can give you this information (more on this below). Plating.... lacZ encodes the enzyme 𝛽-galactosidase which can hydrolyse lactose. Fortunately, for the lab researcher... will just see the vector backbone band. Or, you can choose an enzyme that cuts once in the plasmid backbone...one band. These are only a few examples of how you can use restriction digest to screen your clones, but...using restriction digests to analyze plasmids, which can help you visualize this process, or visit our restriction...restriction analysis blog post. Colony PCR Colony PCR can detect the presence or absence of DNA using lysed...