... multiplexing. pCri plasmids are divided into two sets, each of which includes plasmids with different... As reviewed in our Plasmids 101 series, proteins are overexpressed from a plasmid construct, most often...C-terminal. Each set of plasmids uses the same restriction enzymes for cloning (see schematic below), ...on the Addgene Blog Plasmids 101: E. coli strains for protein expression Plasmids 101: Protein tags ... introduction to pCri, please see the original publication cited below. pCri plasmids are available either...on these and other tags used in the pCri system, please see Plasmids 101: Protein Tags. Beyond E. coli...Bacillus subtilis and Pichia pastoris pCri also includes plasmids designed for protein expression in systems...

... long it takes to make a plasmid? Or how much time you have to spend cloning before you can start your...cost required to make a plasmid. In 2023 (and at closer to 2 million plasmids shared than not!), we think...great Plasmid Debate of ’18, Blue Flame depositor John Doench calculated the time to make a plasmid. Doench...simple insertion of a PCR product into a vector. The time it takes to make a plasmid could increase dramatically... make a plasmid? Doench also calculated the cost of consumable reagents used to make a plasmid, which ... to make plasmids that already exist, and we significantly reduce costs of producing plasmids with high-throughput...lot of plasmids, it’s almost always faster to order from us!) Ask the PI If you want a plasmid for free...

...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...sequence. This means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic ...background via P1 transduction. Plasmid Expressing lambda red genes from a plasmid allows for a mobile recombineering...recombinant clones are generated.  A simple way to do this is to express lambda red genes from a plasmid with...desired modification. Colony PCR can be used to screen for positive clones in most cases, and restriction... is an alternative method that can be used for cloning or genome engineering and is based on homologous... flexibility to modify the E. coli chromosome, plasmid DNA or BAC DNA.  To use the lambda red recombineering...

...conventional PCR. Biallelically disrupted cells can be easily identified. Even in the case of clones with monoallelic...primer validation, and screening by PCR for biallelic deletion clones:    Like the video? The Orkin...polymorphism analysis, allele-specific PCR, T7EN1 cleavage assay, sequencing, RT-qPCR, or immunoblotting to detect...targeted deletions is to produce monoallelic deletion clones. Cells with just a single intact copy at a given...procedures. The video details how to transfect CRISPR plasmids into MEL cells by electroporation, sort your transfected...paper describes their methods for CRISPR design, cloning, and delivery, as well as their techniques for ... PubMed. More CRISPR Refereces: Browse CRISPR Plasmids CRISPR Information at Addgene More CRISPR-...

...and our new cloning grade DNA service. We've had 11 blog posts on COVID-19 featuring plasmid tools from...we also launched the cloning grade DNA service. This service features 210 plasmids: empty backbones, fluorescent...protein tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning grade DNA to you, we'd ...to Elute and Store Your Plasmid DNA? by Paolo Colombi Should I store my plasmid in TE, Tris, or water?...Digital PCR for AAV Quantitation by Kate Harten DeMaio We made the switch to droplet digital PCR to quantify...Here we cover five methods to do this. 5. Plasmids 101: Plasmid Incompatibility by Leah Schiesow If you're...save you days of time so that you can begin your cloning experiment right away. And without further ado,...

...time spent cloning. In contrast, the short homology required by MMEJ can easily be added via PCR amplification...which facilitates easier cloning. An overview of PITCh. The PITCh plasmid contains a GFP-Puro cassette...cassette via PCR, and this construct is inserted into the PITCh vector via In-Fusion or SLIC cloning. Step 2...sequencing of puromycin resistant clones, they found that 80% and 50% of clones displayed proper insertion at...Guide Pages Find CRISPR Plasmids for Your Experiments Deposit Your New Plasmid Tools with Us  ...Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about...month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction There...

...Restriction Cloning Get Tips on Analyzing DNA Sequencing Results Check out Plasmid Cloning by PCR Resources...replaced by real-time PCR to answer the same experimental questions. Real-time PCR detects and quantifies.... Southern blotting and quantitative real-time PCR Southern blotting, named for its inventor Edward ...applications range from confirming the results of cloning or knockout experiments to forensics and clinical...process, rather than just at the end as in standard PCR. This allows the researcher to compare different ...requires a large amount of high-quality DNA, real-time PCR has several advantages including easier automation... have a lower false positive rate than real-time PCR (4). Restriction mapping and modern sequencing Restriction...

... genes cloned into plasmids to investigate for each of these planarian species. These plasmids all have...), and then use PCR and molecular cloning to insert the cDNA into the desired plasmid backbone. The Sánchez...experiments in planarians with their recent plasmid deposit. These plasmids enable two main types of experiments...dsRNA-induced RNAi experiments. Notably, this plasmid harbors a multi-cloning site flanked by T7 promoters to allow...Students can produce DNA templates via PCR off of the same plasmid and use these to create riboprobes for...they can easily generate off of a circular plasmid via PCR. Typically, to make these constructs, developmental...their own molecular cloning and investigating genes not in this collection, they may clone DNA sequences for...

...Error-prone PCR: This method amplifies the wild-type template with a “sloppy” version of PCR that results...mutagenesis (SDM) PCR, but with a twist since it relies on the sequential nicking, degradation, and PCR-generation...a universal primer is used for this round of PCR. The PCR product is digested with DpnI to remove residual... Can I use my favorite plasmid with this protocol? Maybe. Not all plasmids will work for the one-pot...strands of the plasmid will be nicked during the first BbvCI digest and the whole plasmid will be degraded...those in the plasmid. Again, if they are in the opposite direction, both strands of the plasmid will be nicked...map of your plasmid. If the sites are in opposite directions, you just need to use a plasmid with BbvCI...

...Learn New Plasmid Cloning Techniques Pushing the Limits of DNA Assembly Plasmid Cloning by PCR Resources...vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor ligation for next generation..., Francis. "The ligase chain reaction in a PCR world." PCR Methods Appl 1.1 (1991): 5-16. PubMed PMID:...interest are annealed to target DNA (genomic DNA or a PCR amplified fragment) and treated with a high fidelity... T4 DNA Ligase in applications such as routine cloning or DNA library preparation. However, when a method...Genetic disease detection and DNA amplification using cloned thermostable ligase." Proceedings of the National...PMC50775. 6. Barany, Francis, and David H. Gelfand. "Cloning, overexpression and nucleotide sequence of a thermostable...

...Read our Plasmids 101 blog series Find other plasmid protocols and tips Browse our plasmid cloning blog posts...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... or agarose gel slices Product: plasmids or DNA fragments The plasmid purification and DNA gel extraction...purification Starting material: PCR product or agarose gel slices Product: purified PCR product or DNA fragments...from the column, as detected by qPCR, but this did not reduce cloning efficiencies.  When compared to ...depending on the type of DNA you’re purifying (plasmid, genomic, or DNA fragments from agarose).    In...

... can clone the PCR products into a pBluescript cloning vector and sequence the resulting plasmid. It ... target plasmid and pX330-sgRNA plasmid (Mashiko et al. 2013). The pCAG-EGxxFP target plasmid contains...pCAG-EGXXFP plasmid along with the individual gRNAs in their PX330 plasmids (or similar plasmids for gRNA...can be placed in multi-cloning site (MCS) between EGFP fragments. The pX330 plasmid contains humanized Cas9...sgRNA can be cloned directionally into the BbsI site. (c) The pCAG-EGxxFP target plasmid was co-transfected...insert this region into a pCAG-EGXXFP plasmid using standard cloning techniques (The XX indicates where ...pCAG-EGXXFP plasmid? They are the perfect primer sets to use to genotype your pups. A simple PCR with these...

...plasmids, expression plasmids, gene knock-down plasmids, reporter plasmids, viral plasmids, and genome engineering... of scientists and plasmid lovers - serves as an introduction to plasmids. Plasmids 101 will provide you... chosen based on the plasmid you want to clone into. Regardless, once the cloning steps are complete, ...utilize plasmids across a wide range of applications. Some common plasmid types include cloning plasmids...be asked to design, modify, or construct a plasmid. A plasmid is a small circular piece of DNA found in... components that make up a plasmid and why each is important. Our “Plasmids 101” series designed to educate...advancing the field.   What is a plasmid? At their most basic level, plasmids are small circular pieces of...

...inserted it into a plasmid, creating an α-complementation cloning vector. When a cloning reaction goes to...This post is part of our ongoing Plasmids 101 series. Plasmids 101 will provide you with an overview ...of the Plasmids 101 posts here. Now that we have covered antibiotic selection here at Plasmids 101, we...screening your cloning reaction. Being able to select for colonies that contain your plasmid is a great start... start when cloning, but how about being able to choose those that contain plasmid with an insert? Blue-white...Make sure you use a proper plasmid (i.e. contains the α-complementation cloning MCS): pGEM-T, pUC18 and ...successful cloning and not because the cell failed to take up the α-complementation plasmid. This way ...

...Additionally, Dr. Formosa has deposited six plasmids with a multiple cloning site in place of the epitope tag for...protocol is surprisingly simple, requiring only a PCR product containing the modifying sequence flanked...to the chromosomal site of insertion. The linear PCR product is introduced into the cell by direct transformation...Addgene distributes several ready-to-use, modular plasmids, combining fluorescent tags, epitope tags, protease...without the artifacts sometimes associated with plasmid-based overexpression. Tim Formosa, at the University...selection marker (KanMX, HphMX, or His3MX).  Each PCR product from this collection will yield an insert...Jürg Bähler have deposited a large collection of plasmids for genome modification in yeast developed by ...

...to focus on PCR-based applications (qPCR and ddPCR.) qPCR Quantitative/real-time PCR (qPCR) has been used...Reaction (PCR) Protocol: How to Design a primer  Molecular Biology Reference Molecular Cloning Techniques... courtesy of UPODMG 1516 irivbrro ddPCR  Droplet digital PCR (ddPCR) works by using oil droplets to partition...used for copy number assays for years. qPCR works like regular PCR, just with signaling molecules added ...partition a single sample into thousands of PCR reactions using Taqman chemistry (see image, left, of ... Resources on the Addgene blog   Droplet Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview...Analyzing Copy Number Variation with Droplet Digital PCR. Methods Mol Biol. 2018;1768:143-160. doi: 10.1007...

...template (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion of the destination vector with...as a universal donor plasmid. The CRISPaint toolkit includes several donor plasmids for protein tagging...polymerase chain reaction (PCR)-based system that allows users to generate a plasmid containing a direct fusion...and a second plasmid containing the homology arms and an N- or C-terminal tag. The two PCR reactions are...only be detected by labor-intensive processes like PCR or by evaluating visual phenotypes. Improving C. ...The Jorgensen Lab recently developed a modular plasmid assembly toolkit called SapTrap to improve CRISPR...repair template. SapTrap includes a prebuilt donor plasmid library containing several types of fluorescent...

...dried out and died. The alternative to cloning rings, dilution cloning into 96-well plates, is extremely time...give rise to single clones, and thus many plates must be examined for single clones and then handled. Additionally... to the expense of dilution cloning. Taking a page from bacterial cloning, we reasoned that we should...cell clones with sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from...Tips for using the agarose cloning method Test your agarose temperature. Cloning cells using an agarose overlay...late-arising clones, and making sure we always maintain a backup replicates of each clone in case of contamination...individual cell clones with different expression characteristics from a population of transfected clones. The technique...

...element cloned into the plasmid) and any other important features that differentiate this plasmid from its...sharing plasmids through Addgene is that we provide an added level of confidence in the plasmids we distribute...ensuring plasmid quality is to stay organized. Here at Addgene, we collect data on every plasmid we distribute...restriction sites used during cloning, and most importantly, the predicted full plasmid sequence. Other information...during the cloning, sample handling, or sequence assembly steps. All of our incoming plasmids are sequenced...sequence analysis, plasmid verification, and using NCBI BLAST. Tips for high-throughput plasmid sequencing If...on the Addgene Website View our Plasmid Reference Pages Get Plasmid Backbone Information from Our Vector...

...fragmenting plasmid DNA, PCR product, or chromosome. One of the main causes of degradation during plasmid DNA ... a kit Read our blog posts about plasmid cloning Browse our Plasmids 101 blog posts Resources on Addgene.org...Biology Reference Find protocols on plasmid cloning Find plasmids for your research ...Nowadays, the most common E. coli strains used for plasmid amplification are endA negative, meaning the gene...degradation and which experiments you can use your plasmid.  DNA elution choices: TE, Tris buffer, or water...polymerases. In the situation where you need to use plasmid in TE for any enzymatic reaction, an effective ...choices that companies offer for the elution of plasmid DNA through their kit. The advantage of using a...