We narrowed to 499 results for: plasmid dna

...efficiency of plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and...Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464–471 . https://doi.... to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due to this inefficiency it is crucial for...enzymes. At the same time Cas9n cleaves the opposing DNA strain which results in two DSBs that can be repaired...panel of eGFP reporters to quantify the on-target DNA editing efficiency of APOBEC-Cas9 editisome complexes...circumvents the need for DSBs in monitoring on-target DNA editing efficiency of APOBEC-Cas9 editisome complexes...after being targeted by a cytidine deaminase base DNA editor. Specifically the BFP mutant (BFPH66) contains...

... single stranded DNA (ssDNA) oligo which usually work better than plasmids. For ssDNA oligo design, the...easiest way is to co-transfect several plasmid together, with each plasmid having a targeting spacer cloned ...for PCR and surveyor. Q15: I used DNA polymerase Takara Ex Taq™ DNA Polymerase for my genomic PCR, but...consider using ssDNA oligo as donor template, it's usually more efficient than plasmid donor. Typical ...200ng to 500ng of 293FT genomic DNA as template after performing gDNA purification and have tried conditions...articles (3/12/2014). With so many options for CRISPR plasmid tools and numerous experimental design decisions...with expression, though it is possible for the plasmid to still express without the G. Because the G is...

...number of plasmids found in each cell, but also whether the plasmid gets integrated into the host DNA or is...types of yeast plasmids are defined below: Yeast Integrating plasmids (YIp): These plasmids lack an ORI...our first few Plasmids 101 posts, we focused mainly on the elements required for plasmid maintenance within...integration. Yeast Episomal plasmids (YEp): These are most similar to bacterial plasmids and are considered “...well-defined genetic system, and a highly versatile DNA transformation system.Unlike most other microorganisms...simple gene replacement/mutation. Yeast expression plasmids used in the lab typically contain all the necessary...how the regulation of bacterial ORIs determines plasmid copy number within the bacterial cell. Similarly...

...your expression plasmid from these strains is not recommended as these auxillary plasmids may be isolated...control such as the pLys plasmid, which suppresses basal T7 expression. The pLys plasmid contains a chloramphenicol...arabinose-inducible plasmid will not express in an IPTG induction strain for example, nor will a p15 plasmid be compatible... for More Plasmids 101 Articles?  Find them all here: http://blog.addgene.org/topic/plasmids-101   ...In a previous Plasmids 101 blog, we reviewed the salient features of several popular strains of E. coli...coli for DNA propagation. While great for cloning purposes, these E. coli strains are not usually well...strain? Protein expression from high-copy number plasmids and powerful promoters will greatly exceed that...

...achieved at the level of the DNA construct, where your RNAi-target is fused to the DNA backbone that contains...for mammalian RNAi Browse all plasmids for RNAi Check out Addgene's plasmids and resources for plant biology...biological function in a direct or unbiased way at the DNA, RNA, or protein level. The “correct” choice of tool...Therefore, techniques that function downstream of DNA, such as RNA interference, can reversibly exert their...predominantly used to disrupt function at the level of DNA, thereby limiting CRISPR’s effectiveness at high ...initial considerations, such as the starting material (DNA-based vector or purified RNA) and the initial state...typically hairpin RNA, or artificial microRNA). Using a DNA template for the RNAi trigger is the most prevalent...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...Alicyclobacillus acidoterrestris (AacCas12b) exhibits optimal DNA cleavage at 48 °C, a temperature incompatible to ...at 37 °C but preferentially nicks the non-target DNA strand. The lab then found gain-of-function mutations...paper in Nature Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase...wheat. Read the paper in Genome Biology Find plasmids containing these base editors Spatially and ...Neuroscience Find the AAV-based SaCas9 genome editing plasmids String assembly gRNA cloning (STAgR) String ...

...regions containing the putative tracrRNA into a plasmid and expressed it in an E. coli bacteria strain....References 1. Harrington, Lucas B., et al. "Programmed DNA destruction by miniature CRISPR-Cas14 enzymes." Science...sheet Resources on Addgene.org Find the Cas14 plasmids here Checkout our CRISPR guide page Browse the...indiscriminately cleave ssDNA in trans like Cas13 and Cas12 do for RNA and dsDNA, respectively. However...activated. Cas14 in diagnostics By cutting ssDNA but not dsDNA Cas14 is not a good candidate for a new genome-editing...combination enabling the detection of RNA, dsDNA, and now ssDNA. By comparing the ability of Cas12 and Cas14...and maybe more efficient Cas systems, Jennifer Doudna’s lab analyzed metagenomic datasets to try to determine...

... in E. coli with two plasmids (Figure 3 below): Input Plasmid: The input plasmid (high copy, ColE1 origin...inducible promoter (PPhlF).  Output Plasmid: The output plasmid (single copy, bacterial artificial chromosome...through Addgene. Their plasmid maps and GenBank files can be found on their plasmid pages. All two-input...indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig. 3A pNR64 pNR160 Fig. 4A pNR220...state machines (RSMs), which take on a particular DNA-sequence configuration (state) based on the identity...defined by the sequence within a prescribed region of DNA termed the “register”. Each chemical input to an ...result of their recombination is the excision of the DNA between the sites (Fig. 1B below). Alternatively,...

...assembly. Plasmids Explore the collection and order individual plasmids via the links below: DNA Ligases... ID Plasmid Gene/insert 165503 pOpen-HSlig4 DNA Ligase IV 165514 pOpen-9N7lig 9 Degrees North DNA Ligase...pOpen-Pfulig Pfu DNA Ligase 165535 pOpen-THEAQlig Taq DNA Ligase 165554 pOpen-T4gene30 T4-DNA Ligase 165564...pOpen-ECOligA E. coli DNA Ligase 165569 pOpen-Tthlig Tth DNA Ligase 165574 pOpen-HSlig3 DNA Ligase III Return... to top DNA Polymerases Addgene ID Plasmid Gene/insert 165500 pOpen-9N7polA 9 degrees N-7 DNA polymerase...Tli DNA polymerase (exo-) 165524 pOpen-T4gene43 T4 DNA Polymerase 165532 pOpen-ECOpolA E. coli DNA Polymerase...Synthetic Biology Plasmids MoClo Guide Drew Endy Lab Plasmids Jennifer Molloy Lab Plasmids Blog: Golden Gate...

... the Doudna lab has used Cas12a’s non-specific ssDNA degradation to develop the method termed DNA Endonuclease...specific DNA sequence, such as the Human papilloma virus (HPV) genome, via a crRNA. An ssDNA-fluorescently...signal when the ssDNA is degraded, is added to the reaction. To enhance sensitivity, the DNA is first amplified...quantified to determine the presence and quantity of DNA, RNA or a mutation of interest. Together SHERLOCK... increase the sensitivity of the assay, targeted DNA or RNA from a sample is first amplified using RPA...coupled with T7 transcription to convert amplified DNA to RNA for subsequent detection by Cas13. This amplification...with the ssRNA reporter enables SHERLOCK to detect DNA or RNA with atomolar sensitivity and single base ...

...incorporated into the DNA during DNA synthesis and then diluted with subsequent rounds of DNA synthesis (cell... for panel design. Find streptavidin plasmids and biotin plasmids at Addgene!     Figure 1: Indirect...found within the cytosol/nucleus, or visualizing DNA instead of proteins. Some stains can diffuse through...targets include transcription factors, histones, and DNA repair enzymes. Note that when using the nuclear ...Dye stains are non-antibody-based stains that bind DNA or free amines of proteins. Depending on the application...previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside analogs like bromodeoxyuridine...stains are membrane-permeant or -impermeant and bind DNA or free amines, depending on the application. Paul...

...PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-p2A-ferritin-p2A-mCherry...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...features and applications of a partiular plasmid or set of plasmids. We hope that these articles make it ... you to find and use the plasmids you need. You can find all the hot plasmids from 2016 below. With over...over 45,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...alone involves much literature searching, DNA synthesis, and plasmid assembly. Luckily, researchers from Douglas...

...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids Topics: CRISPR, Other CRISPR... where we highlight a few of the newest CRISPR plasmids available at Addgene. If you want to read more...will cover: Miniature CRISPR-Cas systems SEND plasmids Prime editing improvements Miniature CRISPR-Cas...scaffold gRNA and making strategic mutations in the DNA binding cavity of Cas12f. The CasMINI system is less...Read the paper in Molecular Cell Find the CasMINI plasmids Efficient CRISPR editing with a hypercompact ...paper in Nature Biotechnology Find the Cas12f-GE plasmids Programmed genome editing by a miniature CRISPR-Cas12f...cells using various delivery methods, including plasmid, ribonucleoprotein, and AAV.  Read the paper in...

... usage bias in Plasmids 101: Codon usage bias Read more about plasmids in our Plasmids 101 blog series...without altering the sequence of the protein. As DNA synthesis has become quite cost-effective it seems...seems easy for scientists to re-synthesize DNA with the most optimized sequence. Unfortunately, it is not...algorithms/resources (Athey et al., 2017) and many DNA synthesis companies, such as IDT, Genewiz and GenScript...available online to both determine and synthesize DNA sequences for optimal expression in your system of... (2000) Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic...Reference Read about genetic code expansion Browse our plasmid collection page  ...

...transforming both fusion plasmids, or mating two single haploid yeast fusion plasmid transformants into a ... in the cell, including protein-protein, protein-DNA and protein-RNA interactions. The 1980s saw a flurry...format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation...reporter system (e.g. the Gateway-compatible bait Y2H plasmid pEZY202  from Yu-Zhu Zhang’s lab) is an example...interaction detection methods (6). In order to bind DNA and activate a reporter gene in eukaryotic cells,...Keegan, L., Gill, G., and Ptashne, M. Separation of DNA binding from the transcription-activating function...eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell. 1985...

...you will also need to deliver a donor plasmid or single-stranded DNA oligonucleotide, so you will need to...editing? Do you want to deliver the components as DNA, or would mRNA or protein delivery be more suitable...transduction or injection as either protein, RNA, or DNA. Finally, once you have identified the best expression...generate stable cell lines. Packaging and Envelope plasmids provide the necessary components to make lentiviral...for in vivo viral delivery. Cas9 mRNA and gRNA Plasmids containing gRNA and Cas9 are used in in vitro ...efficiency of CRISPR delivery. There are a few plasmids at Addgene that have been published as CRISPR ...genome target sequence into EGFP reporter. Target DNA reporter system: Can evaluate component delivery ...

...repository for DNA-based materials, including our popular kits and libraries. This DNA-based materials...trying.  When we started in 2004, we were the plasmid repository. In 2016, we added viral vectors, and...addgene.org! Fun fact! Did you know you can search for plasmids from a publication by pasting the paper's title...Our first request was on September 8, 2004 (for pcDNA-p160MBP, if you're curious.) Since then, we've distributed...

...Episomal vectors- extrachromosomally maintained DNA plasmids that express reprogramming factors To date,...viral origin of DNA replication, OriP and the EBNA-1 sequence that encodes for the DNA binding protein...and to model diseases in vitro. Find stem cell plasmids at Addgene Many sources of adult somatic cells... amplification of the episomes concurrently with DNA amplification in the host cell. This enables relatively...group is an optimized mixture of three episomal plasmids (Table 1) encoding the reprogramming factors. ...enhance the reprogramming efficiency, an extra plasmid, pCXWB-EBNA-1 is used in the reprogramming cocktail...increases protein expression from the other episomal plasmids. When used on PBMCs, the pCXLE episomal vector...

...inhibiting crRNA loading, inhibiting DNA binding, or inhibiting DNA cleavage.   Five ways to anti-CRISPR...Bondy-Denomy J, Corn JE, Doudna JA (2017) Disabling Cas9 by an anti-CRISPR DNA mimic. Sci Adv 3:e1701620...proteins can also be used to prevent dCas9 tethering to DNA. Since these early anti-CRISPR studies, scientists...block CRISPR activity one of three ways: Inhibit DNA binding Example:  AcrIIA4 blocks Cas9’s interaction...crRNA-Cas complex (Zhu et al., 2019). Inhibiting DNA cleavage Example: AcrIIC1 binds to the HNH endonuclease...endonuclease domain of Cas9 and prevents target DNA cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR...CRISPR   Resources on Addgene.org Find CRISPR Plasmids for Your Research Interested in light-inducible...

...Read blog posts related to plasmid protocols Find protocols for purifying DNA without a kit Resources ...precipitate RNA, but not DNA and proteins. Lastly, using a DNase (there are several DNase enzyme products on...As with DNA isolation, scientists commonly rely on RNA isolation kits to make their life easier. Recently...Recently, we published a blog on DNA purification without a kit that outlined several reasons why doing ... one should take care whenever doing any type of DNA or RNA purification to avoid contamination, take ...RNA extraction. RNA is inherently not as stable as DNA- it is single stranded and its ribose groups are ...There are many alternative methods for isolating DNA without a kit. However, that isn’t the case for RNA...