...blog Addgene's Tips for Plasmid Quality Control Plasmids 101: In-house NGS Plasmid Quality Control Now at...sequencing all incoming plasmids. If you are interested in requesting one of these plasmids, you can request ...data for each of their plasmids, from descriptions of key components of the plasmids to partial, full, or...to ensure all plasmids pass our high-quality standards and contain the expected plasmid features.  ...Once Addgene receives deposited plasmids, they undergo complete plasmid sequencing using an in-house Illumina...will compare the NGS results to the plasmid information, other plasmids in the deposit, NCBI sequence and...results and deposited sequences and plasmid information.  For each plasmid deposited at Addgene, QC scientists...

...multiple gRNAs on the same plasmid, you’ll make sure that each cell that gets the plasmid contains all of the...rectangles) are cloned into various plasmids using oligonucleotides. These plasmids contain Type IIS restriction...different purposes.   Gersbach Lab multiplexing plasmids: This plasmid set allows you to express 2-4 gRNAs, with...gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is ...assembled to produce a plasmid encoding the gRNAs and Cas9. As with the Gersbach lab plasmids, multiple Cas9 ...Golden Gate/Gibson Assembly Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through...Golden Gate/Gibson Assembly Multiplexing Plasmids: These plasmids can be used to express up to 8 gRNAs after...

...BioRender.com.   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems...in archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...began building optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be...functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector and are likely the class most... your own research! Classification Structure Principles of CRISPR classification While CRISPR systems... analysis, CRISPR classifications were placed into a fairly simple hierarchy: class to type to subtype...hierarchy, the main aspects of each class and type are easy to understand. Classes are based on the main Cas effector...

...mutagenesis of Plasmids. A) Generation of mutagenized plasmids. PCR primers (green) amplify the plasmid template...with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are isolated from the resulting colonies...the final circular plasmid product pictured here. B) Screening of recovered plasmids for the mutation of...Smaller plasmids (~3 kb) are generally more efficiently amplified than larger constructs, but plasmids as ...DpnI) to eliminate the parent plasmid from the PCR products, the plasmid template should be isolated from...have taken up the mutagenized plasmid. Note that any residual parental plasmid (usually from incomplete DpnI...additional restriction site into your plasmid, then, when the plasmid is digested with the appropriate restriction...

...cells to retain plasmids - if a cell does not inherit plasmid due to improper plasmid partitioning, it...unstabilized plasmids were retained in less than 10% of cells while plasmids containing the plasmid addiction...ensure that plasmids are passed down through generations (Figure 1). These so called “plasmid addiction ...only survive if they retain the plasmid. Since their discovery, plasmid addiction systems have been used...found that if a mini-F plasmid carried a specific 700 bp segment of DNA, the plasmid was propagated better...selection in one plasmid, they were able to create an efficient cloning tool. The plasmid pKSCTMF contains...restriction-modification systems onto plasmids allowed cell populations to retain more of these plasmids as compared to those...

...these CRISPR plasmids (and libraries)? We wanted to know, so we classified CRISPR plasmids containing a... (A) Cumulative CRISPR plasmids shipped by year. (B) Cumulative CRISPR plasmids deposited with Addgene...,000 CRISPR plasmids in our repository, and we have shared almost 300,000 CRISPR plasmids and 12,000 CRISPR...CRISPR plasmids requested each year by year of deposit (Figure 3), we saw that once CRISPR plasmids become... CRISPR plasmids seem to never truly go out of style. Instead, an almost even mix of plasmids (by age)...Total CRISPR plasmids deposited by country; square size is proportional to number of plasmids deposited ...Total CRISPR plasmids requested by country; square size is proportional to the number of plasmids requested...

... Read our Plasmids 101 blog series Find other plasmid protocols and tips Browse our plasmid cloning blog...alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-...kit-free plasmid miniprep protocol from Addgene follows a similar workflow as a column-based plasmid extraction...the DNA is ready for use! Plasmid purification and DNA gel extraction with glass syringe filters Starting... or agarose gel slices Product: plasmids or DNA fragments The plasmid purification and DNA gel extraction...columns, it’s bound to glass syringe filters. Normally DNA does not bind silica or glass, but the addition ...coaxes it into sticking to the hydrophobic glass filter. Each glass filter can capture up to 150 ug of DNA...

...curating plasmid collections. Addgene's "How to cite this plasmid" section Anytime you use a plasmid from ...variety of plasmids in the repository. And last, but certainly not least, we often feature new plasmid technologies...process, we explain how to cite plasmids from Addgene right on our plasmid pages and provide a citation ...comparing two plasmids, we recommend reviewing the cited papers to determine whether the plasmid has been ...collection or if you think a particular plasmid or type of plasmid is missing from our collections pages...spotlight some of the plasmids that have come through our doors in our “Hot Plasmids” section. We’ve also... added the Plasmid of the Day feature to our home page - each day we showcase a new plasmid that you might...

...within the plasmid backbone to generate R-mAbs of different subclasses, including subclasses that were ...important collection.  Creating the Plasmids To begin constructing the plasmids for expression of mAbs, the Trimmer...expression plasmids were DNA sequenced to determine the VH and VL sequence. These plasmids have been deposited...scientific community with the plasmid sequences available from each plasmid webpage. Importantly, the methods...different subclasses, but there are also secondary antibodies that are specific to each subclass. Approximately...mouse mAbs are IgG1 subclass (Manning et al. 2012), which limits the use of subclass specific secondary...original hybridoma. By using subclass specific secondary antibodies and subclassed switched R-mAbs, multiple...

...associated plasmids with Addgene. Since then, Addgene has filled over 6,500 requests for these plasmids. While...without plasmids at Addgene To find out whether scientific articles associated with Addgene plasmid deposits...those containing plasmids that the authors deposited with Addgene and those without plasmids deposited with...2,596 of which had plasmids deposited with Addgene. Articles with Addgene plasmids get more citations ...with plasmids deposited at Addgene – typically about four times as many as papers without plasmids deposited...Papers without Addgene Plasmids in Journal (E) Percent of Papers with Addgene Plasmids in Journal (F) Weight...more interestingly, could we tell if depositing a plasmid with Addgene causes a paper to get cited more? ...