We narrowed to 803 results for: Cre

...libraries here at Addgene, including cDNA, barcoding, screening, and gRNA libraries. Most of the pooled libraries...determined, larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell...library. These cells can then be used for genetic screening, followed by sequencing and further data analysis...some of your plasmids didn’t amplify after your screen is completed!  Producing Your Lentiviral Library... any issues.  Keep in mind that a low MOI will increase the chances of a cell being infected by only one... one viral particle, but it also increases the chances that a cell doesn’t get infected at all. To make...as seeding density and reagent concentration.  Screening time Phew! You are now (finally!) ready to use...

...pooled libraries have been created in lentiviral systems for mammalian screens, which enables researchers... as the screen readout. This retroviral mouse CRISPR pooled library can be used for screening of cells...pooled libraries are popular tools for genetic screens, enabling genome-wide interrogation of the genes...required for cellular processes. For a successful screen, it is necessary to identify the gRNAs that elicit...are poorly transduced by lentivirus, and thus screening with available CRISPR libraries was not possible...solve this issue, Sarah Teichmann and colleagues created a retroviral CRISPR knockout library using gRNAs... from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation and activation...

...goals and the resources. Instead of creating a start up budget, create a start up list.  In this post we...award by giving you step by step instructions on creating a budget, making decisions based on that budget...budget, and negotiating in regards to the budget. Creating a budget We start by using your research specific...budgetary needs from your experimental goals. When creating your budget, you should take the following steps... that timeline Avoid writing down prices as you create your list. This helps to ensure that all of the...”, “colony”, etc. This will help save time by screening out irrelevant candidates, and, more importantly...doesn’t have to be complicated, or difficult to create this list. Start BEFORE searching for a faculty...

...gene expression. By itself, increasing the copy number of a gene increases its expression. However, if...as plasmid copy number increases, the amount of repressor protein also increases thereby intensifying repression...When they increased concentrations of IPTG, a constitutive promoter upstream of GFP increased expression...that is responsive to a repressor protein, the increase in expression due to copy number is kept in check...origins of replication, the stabilized promoters created by Segall-Shapiro et al. showed nearly no position-dependent...stationary phase, the number of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a...compared to a constitutive promoter. To simulate the increase in plasmid copy number during the transition to...

... edited. Created with BioRender.com. Add silent mutations near point mutations to create 3-base (...target can increase the odds of incorrect editing outcomes, especially when MMR is inhibited. Created with ...edit. Figure 1: Prime Editing schematic. Created with BioRender.com. The pegRNA specifies both...PBS) and reverse transcriptase template (RTT). Created with BioRender.com. The first base of the ...to Cas9 with canonical sgRNA structure (right). Created with BioRender.com. What to edit The sequence... mismatched bases can often evade MMR (right). Created with BioRender.com. For specific prime editing...nicking sgRNAs ~50 bp from the prime editing nick. Created with BioRender.com. PE3b and PE5b: In this...

...activation. Created with BioRender.com. Tet1 demethylase Ronggui Hu’s lab has created pdCas9-Tet1-...targeted demethylation is associated with increased transcription. Created with BioRender.com. Tet1 initiates...the catalytic domain of p300 acetyltransferase increases levels of H3K27ac histone modification at specified...This plasmid is used with pcDNA3.1-MS2-Tet1-CD to decrease methylation and activate transcription. A lentiviral...DNA oxidation and repair factors that allow for increased gene activation at the targeted site compared ... of transduced cells. Grant Challen’s lab also created constitutive (pCMV-dCas9-D3A) and Tet-dependent...is associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase MQ1...

...closer to an innovative resolution. Creative negotiation can also decrease or entirely eliminate the feeling...and everyone leaves unhappy.   Negotiation as a creative problem-solving process Although at first glance...negotiating, what if we instead approached it as a creative problem-solving exercise? Asking questions like...involved and be used as a guide to point you toward a creative solution where everyone wins. What are the underlying...but still be beneficial to both.  Coming up with creative solutions where everyone wins can initially feel...person. It can especially be difficult to think creatively when you are in the middle of a frustrating situation...practice will make negotiating feel more natural. Creative negotiation skills will help you strengthen relationships...

...engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest...sequence preferences must largely be ignored. Created with BioRender.com. The hammer: Gene knockout...terminus of the protein, to maximize the chances of creating a non-functional allele. For a 1 kilobase gene...primary importance (more on this below). Created with BioRender.com. The jigsaw: Editing by ...the need for single cell cloning and subsequent screening for successful edits. This is a very time consuming...constrained by the desired location of edit; efficiency decreases dramatically when the cut site is >30nt from the...options that can greatly expand gRNA options. Created with BioRender.com. Two newer technologies ...

...of themselves. This increases the number of copies of the TE while also increasing the size of their host...mutagenesis screens that detect loss-of-function or gain-of-function mutations. In these screens, the transposons...transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best for screening promoters and enhancers...Then they can be inserted into new target sites. Created in BioRender.com.   Class II TEs: DNA transposons...which produces a target site duplication (TSD). Created in BioRender.com.   Autonomous vs. non-autonomous...capacity >100 kB, although integration efficiency decreases with cargo size. Sleeping Beauty has a close-to-random...can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum cargo capacity of ~...

...round, due to the decrease in ligation efficiency. To combat this, you can increase the volume of competent...other independently regulated transcription units. Created with BioRender.com.   Planning makes perfect... to any tags or fluorescent markers? If you’re creating a multigene construct, in what position do you...stop codon; TAG, C-terminal tag; TER, terminator. Created with BioRender.com. The Sainsbury Laboratory...as well as the restriction enzymes, and it will create the resulting plasmid map and identify any mismatches... new bacterial cultures from your kit plate and create your own glycerol stocks for more frequent use....the MoClo assembly levels, you will be using an increasing number of parts in your one-pot one-step reactions...