...Zetsche, B., Gootenberg, J., Abudayyeh, O., Slaymaker, I., Makarova, K., Essletzbichler, P.,  Zhang, F. (2015... diagram below for an example of how this works. Make sure to order 5’ phosphorylated oligos or treat ...single plasmids are generally more efficient. Pro-Tip Make sure to reverse the orientation of the crRNA’s direct...

... task will keep you from repeating your work or making similar mistakes in the future. Stay organized...camelCaseFormat since spaces between each word can make it difficult to accurately call a file. Version...your exact changes. Alternatively, you can simply make a note in your methods section about the edit that... Your Experiments Try out the CRISPR Software Matchmaker Additional Resources on Addgene.org Find Protocols...

...behind. While commercial kits are available for making small point mutations, large deletions/insertions...Current SDM methods rely on in vitro assembly. To make a point mutation, you design two oligonucleotide...instead of relying on in vitro assembly, they could make the E. coli do the work by using the principles ...-12 kb range. REPLACR-mutagenesis also requires making your own electrocompetent cells in which the phage...

...between blue and white colonies. Take care in making your plates: X-gal is light and temperature sensitive...autoclaving. If spread on top of pre-made plates, make sure it is evenly distributed and allow sufficient...specific analysis, like PCR or restriction digest. Make sure you use a proper E. coli strain (i.e. contains...JM109, STBL4, JM110, and Top10 are a few examples. Make sure you use a proper plasmid (i.e. contains the...

...how the new kit works, the research the kit now makes possible, and how it compares to his CRISPR/Cas9...step. The kit also has other unique aspects that make it especially well-suited for use in stem cells....hands of pretty much all investigators. Our kit makes it extremely simple to design TALENs. It only takes...apples-to-apples comparison of all those mutations. If you make induced pluripotent stem (iPS) cells from patients...

...properties of the fluorophores you are considering to make sure that they’ll be compatible with your instruments...properties allow for efficient signal amplification, making biotin conjugated antibodies good options for detecting...antibody. 2) Endogenous cysteines, such as those that make up the disulfide bonds between antibody chains. ...antibodies yourself, there are kits available to make the process quick and easy. Before getting started...

...scientists commonly rely on RNA isolation kits to make their life easier. Recently, we published a blog...cold and work quickly to mitigate RNA degradation. Make sure to use DEPC-treated or RNAse free water. If..., 2006).  View our protocol for RNA extraction! Making the simple protocol even more foolproof (still ... if you need really clean RNA, it’s important to make sure that when extracting, to only take the aqueous...

... see who you are when you look at their profile. Make your professional contact information available.... be important for a good friend, but if you are making a professional inquiry, you always want to read...type of connection can help you gather information, make introductions, and give advice for your interviews...should be as good or better than your resume Don’t make the mistake of having a shoddy profile and not updating...

...experiments, and here we will focus on a unique gene that makes the popular GatewayTM method possible: ccdB. But... more about it? Read on to find out how ccdB can make your cloning experiments a little easier. One of...contain your insert of interest. Simply stated, ccdB makes cloning easier by selecting against vectors that...not contain the recombinant plasmid and therefore makes the cloning process much more efficient, as one ...

...address the research challenge. Now is the time to make bold statements. While some of the statements may...6-12 months to give feedback on the application. Make sure to invest time and consider the reviewer's ...the next application. Although some comments may make it clear that the reviewer misunderstood the point...that was being made, take it as an opportunity to make clearer statements on the next round of applications...

...or a G to A change on the opposite strand). ABEs make an A to G change (or a T to C change on the opposite...catalytically dead or nickase) fused to a deaminase that makes the edit A gRNA targeting Cas to a specific locus...efficiency for mammalian edits is to ensure the editors make it into the nucleus and that they are expressed ... 98-99% target modification in primary T cells, making them a promising tool for cell therapy applications.... J., Murray, R., Packer, M. S., Rinaldi, C., Slaymaker, I. M., Yen, J., Young, L. E., & Ciaramella, G...

...for antibodies. Recombinant antibody technology makes it easy for scientists to share, remix, store, and...     How are Recombinant Antibodies Made? To make recombinant antibodies, you first need to know the...  Second, plasmids are easy to store and share, making recombinant antibodies the most practical choice... encoding antibodies, scientists will be able to make higher affinity antibodies, modify antibody function...

...changes (Figure 1), but it can be challenging to make these base changes, especially in primary human ....20-m to edit primary human T cells efficiently makes it a promising tool for T cell therapy development...robust editing in human cell lines and primary cells makes them a great addition to the base editing toolbox..., Liquori AJ, Murray R, Packer MS, Rinaldi C, Slaymaker IM, Yen J, Young LE, Ciaramella G (2020) Directed...

...Tweeting and texting, actually writing an e-mail or making a phone call is a big commitment when you’re busy... to consider before you commit: Double check to make sure the answer to your question didn’t come with...expedite and improve your customer service experience: Make sure you’re contacting the correct company. This...as possible about the issue. For Addgene support, make sure to mention the plasmid catalog number, or at...

... jobs needed to be done (Who is responsible for making the running buffer this week?). Scientists negotiate...were hoping that your lab mate would have time to make comments on your manuscript. If you give her this...practice and hone your skills. This practice will make negotiating feel more natural. Creative negotiation...the person/team for help? What can I offer them to make it a win-win situation instead of just extra work...

...Jacob Corn contacted us in 2014 to ask if we could make a model of Cas9 based on the structure published...Jennifer Doudna’s new book A Crack in Creation. Making models more useful through flexibility It is common...the several Lys/Ala mutations reported by Ian Slaymaker and colleagues to reduce off-target hits in a ...our hands-on teaching tools that are designed to make the molecular world real for students. Many thanks...

...limiting reagent for my experiments, so I always make more AAV than I think I need. Unlike lentivirus,.... If you’re new to creating gradients, practice making the iodixanol layers before doing your first purification...purified and buffer exchanged virus, remember to make one or two small aliquots to use for qPCR titering...Additional resources on the Addgene blog Find out what makes AAV a versatile tool for gene expression in mammals...

...to help make the most of these pages, we've written this post to guide you through them and make the best...can be found in this location as well. These maps make it easy for scientists to see if the plasmid would...their plasmids are used in a publication. Addgene makes this easy by providing a template citation for you...

...more CRISPR tools are being published each month, making CRISPR a great choice for your next experiment!...control gene expression. Base editors can help you make precise point mutations near the gRNA target site...single-stranded DNA oligonucleotide, so you will need to make sure your expression system and delivery methods...Envelope plasmids provide the necessary components to make lentiviral particles. Stable, tunable expression...

...applications, and the quality of a standard curve can make or break an experiment. Here, we will provide an...the concentration of the unknown sample.  How do I make the standards for a standard curve? You create a...with BioRender.com.   When choosing a standard, make sure that the sample is pure and free of any contaminants...start with a higher concentration of standard or make smaller dilutions.        As mentioned above, it...