...mammalian cells (Cong et al ., Science 2013, Ran et al ., Cell 2013, Ran et al ., Nature Protocols 2013). For ...codon-optimized SpCas9; sgRNA can be co-transfected as PCR amplicons (Ran et al., Nature Protocols 2013). Individual...sgRNA can be co-transfected as PCR amplicons using a protocol similar to SpCas9 sgRNA PCR (Ran et al., Nature...and its single guide RNA (sgRNA) into one plasmid (Ran et al., Nature 2015). The dual vector system uses...Genome Engineering using CRISPR/Cas Systems. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu... Cas9 nucleases. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X,...CRISPR Cas9 for enhanced genome editing specificity. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino...

...CRISPR-associated transposases (CASTs) combine the large-scale capacity of transposases (Tns) with the precise...Find plasmids for CRISPR-associated transposases (CASTs) for insertion of donor DNA. CRISPR...CRISPR Plasmids: CRISPR Transposases (CASTs) Browse CRISPR Plasmids By Function Genome Editing Cut Base...Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting...lacks nuclease activity, instead recruiting the transposase (comprised of multiple components) to target ...locations in the genome defined by a guide RNA. The transposase can then integrate donor DNA. Editing by CASTs...CASTs is not considered "scarless", as the transposon homology sequences are integrated along with the donor...

...CRISPR plasmids that create double strand breaks in DNA....Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...CRISPR nickase plasmids for the generation of single-strand breaks. CRISPR...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...

...including transformation, transduction, conjugation, and transfection. Transformation, transduction, and conjugation...created phages. Transfer of this DNA to another cell is referred to as transduction. Transferred DNA once inside...different methods of DNA insertion. Transformation Transformation is the uptake of genetic material from... to cells, to increase cell membrane permeability and thus transformation efficiency. Check out Addgene...fluoresce green. Comic by Maya Kostman.   Transduction Transduction occurs when foreign DNA or RNA is introduced...from a ‘helper’ phage prior to transduction. Scientists also use transduction to introduce foreign DNA into...donor can transfer genetic material to the recipient bacterium. The genetic material transferred is commonly...

... plant scientists do transient leaf transformations The development of transgenic plants is a common problem...that the transformation and gene expression can be tracked. During a transient leaf transformation, the engineered...method. Transfer your plant into the dark 16 hours before the transformation. We routinely transfer our plants...developing transgenic hairy root lines in tissue cultures takes half a year, and generating a transgenic plant...plant can take even longer, a transient plant leaf transformation process could save the plant biologist...development of transgenic hairy roots takes at least six months. Generating a transgenic plant takes even...reproducible. Prior to creating transgenic cultures or plants, our transient expression method enables us...

...Associates, Inc., a national grant consulting firm, and Smartegrants, a grant professional development firm... the field of grants. What is a grant professional?  In the past 5-10 years, the Grants Profession has...readiness, identify grant opportunities, secure grant awards, and manage grant projects and funds. You will... for grants, the amount of grant funds available and the increasing regulatory process for grants has ...become a grant professional? Employment opportunities for those with expertise in grant writing, grant management...as a Grant Professional to mentor and coach you. There are many grant consultants who offer grant training...including Smartegrants, to help those entering the Grants Profession. Whether you’re transitioning from a...

.... This is why they are called transferable skills. Why are transferable skills so important? It is important...   What is a transferable skill? Time management. I needed it when balancing a handful of demanding courses...through school, jobs, and volunteer work, and I guarantee you have developed them through similar experiences...useful anywhere I work in the future; they are transferrable across most, if not all, industries and work...important to identify which transferable skills you possess because any potential employers, collaborators...to do the job. You may think this means you’re guaranteed to get the job, however, many times the hiring... examine not just technical skills, but also transferable skills, before offering someone their dream ...

...Nature . 550(7676):407-410. PMID: 28931002 Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...CRISPR-Cas9. Cell . 167(7):1829-1838. PMID: 27984730 Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...binds, Cas9 separates the double stranded DNA target and cleaves both strands near the PAM. (6) The crRNA:...: dCas9 fused to transcriptional activators or repressors can modulate transcription in a reversible manner...palindromic repeat sequences. (1) The CRISPR array is transcribed to make the pre-CRISPR RNA (pre-crRNA). (2) The...is processed into individual crRNAs by a special trans-activating crRNA (tracrRNA) with homology to the...:tracrRNA:Cas9 complex unbinds after the double-strand break. CRISPR systems, found in 95% of archaeal...

...feel they lack formal training in a variety of transferable skills. At Addgene, we've set out to fill this... by highlighting that researchers learn many transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Negotiation. As a grad student...I was negotiating with my PI on how to spend my grant money (Can I attend this conference?) and with my...Hawaii?). So how can you approach improving this transferable skill? Negotiation as a zero sum game Some ...is the 6th year grad student frustrated that the brand new grad student keeps using the machine? Let...negotiation skills You can easily work on this transferable skill without any formal training or coursework...

... which is ease of transformation. There are many motivations to generate transgenic Arabidopsis, from ...from studying transcriptional and translational dynamics of genes and proteins in living plants, to complementing... dip transformation method. The general steps for this method include: Cloning and transforming a plasmid...integrates transfer DNA (tDNA) into the genomes of the plants it attacks Growing the transformed agrobacterium...obtain optimum transformation efficiency. It can take 6-8 weeks from the date of transformation to having ...2013). Preparing plants for transformation Whatever plants you are transforming into will be your T0 generation... ready to transform when they begin flowering. It is important to perform the transformation not long ...

...improve your grant writing and hopefully increase your success rate. 1. Submit your grant early! First...in writing small travel grants to attend conferences and a seed funding grant for a proof-of-principal...www.assaygenie.com. No matter what facet of academia you are in, grant writing can be the lynch pin that results in your...to be much use. 2. Originality is essential for grant success The two most important things reviewers ...sure that comes across. Check out some in-depth grant writing tips at the Human Frontier Science Program's...Skills 5. Practice makes perfect, apply for small grants too There are actually quite a lot of different... researchers, so look into applying for smaller grants from within your institute or a private funding...

...Blog Transferable Skills Guide: Problem-Solving Transferable Skills Guide: Teamwork Transferable Skills...feel they lack formal training in a variety of transferable skills. At Addgene we're helping to fill this...both helping researchers recognize their MANY transferable skills learned while working in the lab and ...Today, we’re highlighting an oft-underlooked transferable skill: creativity. I’ve heard quite a few scientists..., it’s clear that creativity is absolutely a transferable skill developed during PhD training. Creativity...problem. In a study outlined in Originals by Adam Grant, a creativity expert at Stanford conducted an experiment... anywhere! References and Resources References Grant, A. (2017). Originals how non-conformists move the...

... generate and costly to share. What is a RANbody? A RANbody is a fusion between a reporter and a nanobody...nanobody sequence could be used to make a RANbody. RANbodies are expressed in 293T cells and are secreted...smaller size of RANbodies compared to antibodies. Staining with multiple HRP-RANbodies is indistinguishable...HRP-conjugated RANbody can be used at a time. Samples can be sequentially stained with two HRP RANbodies if the...Chicken IgY RANbodies are easily detected with a labeled anti-chicken antibody. Chicken IgY RANbody detection...you ready to start making your own RANbodies? Great! All of the RANbody plasmids from Yamagata et al can...nanobodies to turn into RANbodies. If you know of a nanobody that would make a useful RANbody, consider leaving...

...Chiara, the tech transfer expert who understands the somewhat complicated Material Transfer Agreement process...that they lack formal training in a variety of transferable skills. At Addgene we've set out to fill this... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...usually the products of teams: Watson, Crick, & Franklin, Meselson-Stahl, Hershey-Chase...Who was I to ...Discovery is exactly that: a myth. The original Dr. Frankenstein from Mary Shelley’s novel worked alone, but ...on the Addgene Blog Read other posts in the Transferable Skills Guide Management for Scientists: Managing...

...may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try amplifying the...immediately prior to their transduction has been shown to increase AAV transduction efficiency by approximately...immediately following transfection. In other words, when exchanging the medium after transfecting cells, replace...Fibronectin is another membrane-interacting protein that can be used to enhance transduction efficiency in cells...your transductions aren’t working, you can check if virus is being generated by your transfected packaging...amplifying a viral vector, collecting virus, and transducing target cells, one may have the unfortunate experience...For some unclear reason, cells are not getting transduced. Here, I will share five hail Marys that have...

...triple T’s of biology are transformations, transfections, and transductions! In this blog we will discuss...intricacies of transductions, we've got you covered there too. What is a transduction?  Transduction is the ... experiment. Using transduction vs. another method Transfection and transduction seem to achieve similar...picky when it comes to transfection/transduction methods. Some ‘difficult to transfect’ cells lines are amenable... process of transferring foreign nucleic acid into a cell by a viral agent. Transductions are similar ...to find out more! Molecular basis of transduction Transductions utilize the ‘normal’ mechanism of viral...cell, enters the host nucleus, is transcribed into RNA, and is translated into protein.   Is there an...

... blog Management for ScientistsTransferable Skills Guide: LeadershipTransferable Skills Guide: Teamwork...the right time. Like many of the posts in our Transferable Skills series, this post is not meant to be ... that you could strengthen. The right set of transferable skills, coupled with your technical skills, ... can help, and if and when you find yourself transferring this skill to another job, it will be important...also aggravate long-term personal conflicts.  Transitioning a conflict into a collaboration may not be the...willing and able to solve, don’t take that skill for granted. When you go on interviews or are looking for a...

...feel they lack formal training in a variety of transferable skills. At Addgene, we've set out to fill this... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Problem-Solving. I don’t know ...in my more recent positions outside of the lab. Granted, I currently work on the Scientific Support Team... careers, I always felt that descriptions of transferable skills seemed fuzzy or ill-defined. In this ...your life. This tactic is a good way to see how transferable these skills really are, while also practicing...systematic, logical thinking.  If you are already a lab veteran at this point, you probably have better developed...

...express a transposase or reverse transcriptase so they can transpose. Both class I and II transposons can be...eukaryotic genomes.  Components of a transposon system Transposon: The transposon is the DNA sequence that moves... encodes the proteins the transposon needs for transposition. Transposition protein: This is the protein...themselves as RNA transcripts and then rely on a reverse transcriptase enzyme to reverse transcribe back into ...reverse transcriptase and integrase enzymes which are required for LTR retrotransposon transposition. Non-LTR...reverse transcriptase activities.    Figure 1: Overview of retrotransposon transposition. Retrotransposons...2021. To mobilize a transposon, first the transposase enzyme binds the transposon’s long terminal repeats...