...host cells immediately prior to their transduction has been shown to increase AAV transduction efficiency... such cationic reagent that is commonly used and has been shown to increase transduction efficiency by...single-use aliquots. Never use a polybrene solution that has been thawed more than once. Fibronectin is another...polybrene (e.g., hematopoietic or primary cells), and has been shown to increase transduction efficiency by...

...regulatory elements. To solve this problem, the Zhang lab has previously used SaCas9 (1053 aa/3.2 kb) with AAV ... these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...THE ORIGINAL NgAgo ARTICLE DISCUSSED IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED ...be the best option for HDR, but this possibility has not been experimentally confirmed. Researchers have...

...for the presence of an insert. Traditionally this has been done with restriction enzyme digest; however... Find Empty Backbones at Addgene Each approach has advantages and drawbacks, which are summarized in...insert-specific primers but also tell you whether the insert has the correct directionality. Verifying the insert ...and are more likely to work in a PCR reaction that has bacterial debris. Use a positive control. A good...

...Fluorescence is the emission of light by a substance that has absorbed light. The emitted light is at a longer ...protein Excitation & Emission (ex/em): Each FP has its unique ex/em peak. Therefore, choose FPs that...detect the emission. For example, if your microscope has only two lasers, at 488nm and 561nm, you will not...Some proteins are very dim (e.g. TagRFP657, which has a brightness of 0.1) and this should be taken into...

...attributes (which turned into www.fpvis.org).  That page has been very useful over the years, and when researchers...system that allows users to see when a given page has unmoderated changes and to view previous versions... online collections of FP spectra. Every protein has a page with all the information in the database relating...

...easy. Most of our equipment has been donated by local companies and BosLab has grown into an organization... biologist with a Ph.D. from UNC-Chapel Hill and has extensive experience targeting RNA with small molecules...

...amplified and none are lost. Each Addgene library has its own depositor transformation and amplification...product for next-generation sequencing Once the DNA has been extracted, the representation of the library... success of your amplification. I hope this post has helped clarify some of the important considerations...

... dronpa fusions. With these various designs, Xin has been able to develop photoswitchable Intersectin,...Intersectin, Cdc42, Raf1, Mek1, Mek2, Cdk5, and Cas9. Xin has demonstrated the utility of these FLiPs in a variety... of cells that actively produce the HMOs. Fatima has demonstrated the successful production of both types...

...AAV2 pAAV-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine 121538  AAV5 pOTTC1484 - pAAV SYN1 HA-hM4D(Gi) 121539 ...121539  AAV5 pOTTC1596 - pAAV SYN1 HA-hM3D(Gq) New chemogenetics AAV Plasmid Serotype Name 119741  ...

...birth several weeks previously. While this claim has yet to be independently verified, the watching world...Kingdom’s Nuffield Council. While the Nuffield Council has recently published a report on the social and ethical...considerations, in making sure that the research has been adequately proven to be reasonably safe and ...

...Davis School of Medicine on this grant. Dr. Trimmer has created thousands of high-quality antibodies/nanobodies...mission This is the first NIH grant that Addgene has received. Addgene prides itself on being financially...The timing is ideal as the NIH’s BRAIN Initiative has been funding laboratories, such as Dr. Trimmer’s ...

...process, though the process has changed a lot over the last twenty years! This has helped us develop a fast... request going forward.  After another scientist has placed a request for your plasmid, they must complete...

... to the original paper that everyone in your lab has been referencing for the last 10 years, and everything...communication further up the collective priority list. There has been progress—for example, a number of funders (e.g...joined Elsevier after her postdoctoral studies, and has held various editorial roles on review and primary...

...derived from the same cell type; however, their usage has been limited by technical problems. For instance,...binary systems in which transient protein expression has a long-lasting effect, such as Cre- or Cas9-mediated...effects. Third, protein purification (once a protocol has been established) may be less laborious than viral...

...sequence if necessary. The example plasmid on the right has a total size of 7.3kb, including a 1.2 kb insert....resulting gel image includes a 1kb ladder (lane 1) that has bands ranging from about 500bp to 10kb, with the ...reference point so you can easily assess how far the gel has run. Bonus: The dyes also run at predicted sizes ...

...Griesbeck of the Max Planck Institute for Neurobiology has been working on genetically encoded indicators of...imaging neurons, Griesbeck adds that this color change has a particular advantage for observing events taking...Maarten Merkx of Technische Universiteit Eindhoven has similar interest in FRET-based sensor proteins, particularly...

...booklet. What has been the most surprising thing about becoming a PI? Tom Ellis has a one word answer...prepared me for what a PI without a personal assistant has to deal with. My inbox currently stands at about ...

...means the bacterium is not naturally resistant but has become resistant somehow overtime. One fantastic ...on a plate! Antibiotic resistance can arise, and has arisen, for many different reasons. Exposure to antibacterial...presence of a known concentration of antibiotic that has been impregnated into a paper disk, and observing...resistance; it’s a natural process of life and one which has allowed bacteria to evolve and survive for millennia...

...studying the genetic underpinnings of these processes has been complicated by the lack of precise genetic tools...Simplified in vivo delivery using SPH mice CRISPRa has largely been limited to in vitro settings due to ...can be delivered to the CNS by lentivirus, which has a larger packaging capacity than AAV. Such an approach... or coding regions. Typically, each gene isoform has a distinct transcriptional start site. Because CRISPRa...

... Cas11-like protein, and a guide RNA. The enzyme has dual activities: processing pre-CRISPR RNA into mature...kilobase-sized gap in the gene editing field. The AbuGoot lab has now developed PASTE, which can “drag and drop” kilobases...PASTE is more specific and efficient than HITI and has higher purity of insertion than both HITI and HDR...therapeutic applications. The potential of CRISPR has yet to fully taken advantage of, but exciting new...