...newest CRISPR technologies and their applications can be exhausting. A quick search for "CRISPR", short-hand...concept of the double nickase system is that you can express two different chimeric gRNAs with the Cas9... 'G'. Q3: What is the maximum amount of DNA that can be inserted into the genome using CRISPR/Cas forHomologous... introduction of a mutation into the genome, how can cells with that mutation be selected/screened? A4... were positively transfected. Alternatively, you can use a selection marker to select transfected cells...template has a selection marker such as puromycin, you can (also) select for the positive colonies by puromycin...

...semi-synthetic or fully synthetic compounds. Antibiotics can be categorized based on whether they directly kill...bit of a gray area as some bacteriostatic reagents can kill bacteria when used at high concentrations (and...antibiotic stocks, see Addgene's Reference Page. How else can antibiotics be used in the lab? Historically, antibiotics... for plasmid selection. Additionally, scientists can use the loss of resistance as a marker for successful...Carbenicillin is more stable than Ampicillin and can be used in place of Ampicillin in most applications... Simplify Your Cloning Experiments See Where You Can Find Antibiotic Resistance on Our Plasmid Pages ...

...email to get information if you can help it. Observe as much as you can in person. Rotate in the lab, if...Connie Cepko to learn about her mentoring style. How can I find out if an advisor is a good mentor? You have...for scientists to change labs – do as much as you can to ensure a good fit in advance. When a lab is interviewing... – I am different.”  Unfortunately, a bad mentor can be bad news for anyone. Try to speak to them more...excellent mentors out there. With some good pre-work you can find a lab that will deserve your hard work and an...

...dual nickases to generate a knockout or edit. This can help reduce off-target activity. Deleting a large... multiple genes at once. Using multiplexed gRNAs can target multiple locations in the genome to modify...common question Addgene Senior Scientists receive is: can I express more than one gRNA from a single promoter...plasmid is available from the Bullock lab, and gRNAs can be inserted using Gibson Assembly or SLIC cloning...sequence, creating flanking overhangs. These overhangs can be customized to link together multiple fragments...IIS enzyme, the unique flanking overhang sequences can link together to allow for ordered assembly into ...Lentiviral Expression Systems (MuLE) Kit: This kit can be used to create lentiviral vectors expressing wt...

...cleavage, the fluorescence is no longer quenched and can be visually detected using lateral flow strips. Since...technique Sensitivity: Single molecule per milliliter can be detected – in the zeptomolar range Time: ~1 hour...Cleavage results in fluorescence and this approach can be incorporated into a lateral-flow readout slip ...Protospacer Adjacent Motif) for target cleavage. This test can be performed in two steps for challenging samples...the ssDNA-FQ reporter. The resulting fluorescence can be observed under either no excitation light, UV ...field-ready, Bluetooth-enabled PCR machines that can even be battery operated, made accessible by the ...sequences. Upon binding, changes in fluorescence can be visualised. Another approach was to apply the ...

...was successful in your organism or cell type? You can use DNA sequencing or other molecular cloning techniques...without any off target effects. However, these methods can be labor intensive and quite time consuming.   As...activity.  By using the RFP:GFP ratio, edited cells can be sorted from unedited cells. In an experiment that... once a mutant is identified, the PB transposase can easily and seamlessly remove the integrated GFP reporter...fluorescent protein (RFP), named GEMCherry2, that can rapidly and efficiently evaluate Cas9 cutting sites...genomic region inserted into the N-terminus of mCherry can be targeted by a sgRNA guided Cas9 causing a DSB ...depending on the nucleotides inserted or deleted, can result in an in-frame shift of mCherry and thus expression...

...But base editors aren’t perfect. They may be slow, can only target certain sites, or make only a subset ...with a faster deamination rate so that deamination can happen before Cas homologs let go. The Liu lab developed...editor, a functional gene III product is produced and can infect bacterial cells. If a phage does not deliver... The editing speed dictates which phage variants can propagate faster than the rate of dilution (passage...editor was named ABE8e. ABE8e is not only fast, it can also be multiplexed: when tasked to correct two point...mutations. Together, cytosine and adenine base editors can correct all four of these kinds of point mutations...of ABE8e to the base editing toolbox, scientists can now make these edits much more efficiently and in...

...GPCR signaling can also modify other properties such as endogenous gene expression, which can lead to changes...chemogenetic receptors hijack cell signaling pathways and can lead to such responses as decreased heart rate (Redfern... is important to consider that the same receptor can elicit different responses depending on what cell...manipulation are neurons where chemogenetic receptors can be used to alter excitability by lowering or raising...). While some scientists argue that these issues can be mitigated by using low doses and running appropriate...such as C21, CLZ, Perlapine, and Olanzapine, that can also activate muscarinic-derived DREADDs. A ligand... of off target effects. These synthetic agonists can penetrate the brain and some, such as varenicline...

...link genotype to phenotype that can be used with mutational scanning: ligand binding assays, phage display...saturation mutagenesis Several methods can be used to create mutational scanning libraries. While each of these...saturation mutagenesis as a quick and easy technique that can be used to generate complex libraries of mutant plasmids...thousands of protein variants can be tested in one experiment. There are many functional assays that link... choosing and validating a selection system. You can test the effect of multiple mutations in combination... bases (NNN) at any given codon site of interest can be cloned into an expression vector with Gibson assembly...mutagenesis FAQ What’s the maximum length of codons that can be mutated? 88 codons, or 264 basepairs, was the ...

...competition, where you can get all your burning AAV questions answered.  Interested? You can learn more here ...travel prize in our open data extravaganza!  Anyone can submit data from their CLOVER lab or Addgene AAV ...

...at Harvard University. There are tons of ways you can get involved in science communication. In this second...from SITN, we discuss some of the many things you can do start your own science communication student group... Pint series and organize evens where scientists can grab a beer and chat about their work at a local ...the News, and member of the SITN podcast team. You can follow Vini on twitter @vinidhra.     Amy Gilson ...News, and is a member of the SITN podcast team. You can follow Amy on twitter @aigilson.   Special thanks...

...smartphone apps you can use in the lab. Whether you’re tired of calculating dilutions or you can’t quite remember...chemical in your obscure growth medium, these apps can help make your lab life a bit easier. Since the post...University of Alberta and describes a technique you can use to find phage proteins that bind to your bacterium...phages are viruses that infect bacteria and that can do so with exquisite specificity (check out our interview... dress up as Mr. Incredible, an added bonus. You can find the rest of our September posts in chronological...post highlights a rare disease, Fibrodysplasia ossificans progressiva (a.k.a. FOP), that, while affecting...Chemistry & Small Molecule Fluorophores Addgene's Cancer Collection Pages Connect the Research Community...

... happen before you even receive the product? How can we consider the environmental impact of those processes... waste directly into the oceans.As consumers, we can choose products that are better for the environment...sacrificing performance or quality. To do this, we can use the ACT label, which is an eco-nutrition label...sciences, some of these plastics are high grade and can be washed and reused. This might sound risky in terms... incentives (for example, by grant agencies), we can shift toward a more sustainable one. The math on ...

...should be no secret that one of the best things you can do during your training is meet interesting people...a job. Ask openly to be connected to someone who can help you land that position such as a direct connection...Interviews are best conducted in person. Perhaps you can offer to buy coffee or lunch at a place convenient...and disappointments. These more personal questions can elicit the most valuable insights you will get from...each Informational Interview with the question, “Can you connect me to anyone else you think I should ...5-12 years worth!) with academic scientists who can’t help us learn about the nonacademic sphere where...

...email reminders. These programs can be synced to your online calendar and can be accessed from your computer...Organizing your lab notebooks, protocols, and to do lists can be easier than you think! Here are three tips to ... up out of the top of the notebooks, so that you can easily spot the colors and text. Tip 2: Use electronic...from anywhere, but also allow you to control who can edit the documents and track the revision history...travel, and you can’t remember where you put your flow cytometry protocol. Or maybe you can’t find the molecular...

...Temperature: You can place your reaction on ice if you are planning to transform right away OR you can store the...group from a different DNA strand comes along, it can attack this covalent bond thus joining the two DNA... TOPO Cloning Reaction into Competent Cells: You can use your standard lab protocol for this; however,...and Analyze 10 White or Light Blue Colonies: You can confirm the presence of your insert by PCR, restriction...when the PCR product and vector ligate; however, it can potentially rebind and nick the newly ligated DNA...minutes (incubating the full 30 minutes will not significantly improve the transformation efficiency).  5. ...

...editing tool. However, Cas9’s endonuclease activity can be limited by the need for a specific PAM (protospacer...bind the target sites for editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA...versatility of the engineered SpCas9-NG and its variants can be used to engineer specific sites that were previously...Anion-conducting channelrhodopsins (ChR) are ion channels that can be used to silence the activity of neurons. Earlier...cysteine.  With the introduction of MerMAIDs, neurons can be turned off quickly and with high precision - a... Anchor. Using specifically designed gRNAs, they can then recruit these LADL Anchors to genomic targets...

...getting buy-in when delegating can help ensure successful results.  Can you provide the information and...you are giving this responsibility to someone who can do the job. It is good to delegate so the person ...result in more engaged, successful employees who can accomplish great things.   To read more on this ... excellent blog on delegation tips and practices can be found at the Time Doctor Blog "The Art of Delegation... How Volunteering at a Professional Organization Can Help You Develop Leadership Skils Set Concrete Goals...manager must do. You can’t delegate the giving of feedback or performance review. You can’t delegate tasks ...

...Diagnostic restriction digest Diagnostic digests can be used to confirm the rough structure of the plasmid...features within the plasmid. Restriction analysis can also be used successfully even if you don't have ... Once you have purified plasmid DNA, this method can be done right in your lab in less than a day. Diagnostic...as well as the predicted size of the insert. You can search NCBI for YGOI to find the particular reference...the dyes provide a visual reference point so you can easily assess how far the gel has run. Bonus: The...The dyes also run at predicted sizes so you can estimate how far down the gel your bands have traveled ...

...suitable for both short-and long-lived proteins, and it can even distinguish between protein variants when used...antibody works well. In larger cell populations, you can deliver these components together using electroporation...protein knockdown experiments! The TRIM-Away plasmids can be found here. Clift et al. Cell. 2017. PubMed PMID...which sgRNAs are used with FIRE-Cas9, experiments can be designed to target multiple loci at once. In addition...memory at nearly any locus. The FIRE-Cas9 plasmids can be found here. Braun et al. Nat Communications. ...encoding synthetic yeast transcription factors that can independently control expression of your genes of...progesterone do not affect each other, these systems can be used together. Aranda-Diaz et al additionally ...