... are the details:   Contest 1: Deck the Lab - Classic Edition This edition of the contest is our traditional...loose on the lab. Have fun! Here’s an example from last year’s contest:   🎶Walking in a Winter Wonderlab...Addgenies will vote on the entries in both the classic lab and home editions. We will select up to 3 winners...

...cloning plasmids, expression plasmids, gene knock-down plasmids, reporter plasmids, viral plasmids, and ... of scientists and plasmid lovers - serves as an introduction to plasmids. Plasmids 101 will provide you...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...cloning site. The ease of modifying plasmids and the ability of plasmids to self-replicate within a cell ...exploited for sequence verification of plasmids.   How is a plasmid constructed in the lab? Due to their...

...First, why are plasmids incompatible? Plasmid incompatibility occurs when multiple plasmids within one cell...control plasmid replication of each plasmid using unique replication machinery. However, when plasmids have... one plasmid lost from the cell at a higher rate than the coresident plasmid. Asymmetric plasmid loss ...including one plasmid blocking replication of another and one plasmid outcompeting the other plasmid for replication... copy plasmid and the larger, low copy plasmid is more likely to be lost over time.  Is plasmid incompatibility...develop a plasmid-based tool that is easy to use with other plasmids.  How do you know if your plasmids are ...about plasmid cloning Resources on Addgene.org Download the Plasmids 101 eBook  Browse plasmids at Addgene...

... used for plasmid-based experiments. What are control plasmids? Generally, control plasmids help to ensure...follow an example of a plasmid-based experiment, delineate its requisite control plasmids, and discuss why ...proper control plasmids would address and answer these questions. Types of control plasmids Part of planning...conditions and plasmids should produce a null effect (i.e., no phenomenon is observed). In any plasmid-based experiment... and not some other unintended factor. Control plasmids are used to minimize any effects of the non-independent...variables.  To illustrate the importance of control plasmids, we will take a step beyond discussion in the ...why these control plasmids are critical for the correct design of the experiment.  The Experiment: Knockdown...

...DNA designed in the lab. Specially constructed plasmids can be used to replace genes in yeast, mice, or...gene. Here, we'll describe the techniques and the plasmids used to inactivate specific genes in mammalian...mind when choosing a knockout strategy! Knockout plasmids Homologous recombination is a mechanism to accurately...randomly integrated all or large portions of the plasmid. Deliver your construct to your target cells. After...Cre-mediated recombination.   Knockout/Knock-in plasmids Gene targeting methods also make it possible to... and insert GFP into the genome, we’d create a plasmid similar to the one shown below, where the sequence...endogenous protein with GFP, as seen in blue flame plasmid OCT4-eGFP-PGK-Puro from the Jaenisch lab.   ...

...more cells = more plasmid. Plasmids can vary in copy number, the number of plasmid copies supported within...where your plasmid yield is sub-optimal? If you are consistently getting sub-optimal plasmid yields from... optimize your plasmid prep We'll discuss the following techniques to increase plasmid yield. Keep in ...genotype, as these mutations improve plasmid stability and yield. If a plasmid is prone to recombination Use ... a strain optimized for plasmids with repeat elements, such as viral plasmids with inverted terminal repeats...to reduce plasmid recombination, but are also endA+. endA encodes a thermostable periplasmic endonuclease...co-purify with your plasmid DNA and lead to degradation of your prep. Many plasmid prep kits recommend...

...blastn), Standard Protein BLAST (blastp), and Translated BLAST (blastx). NCBI has a terrific getting-started...your wait time for blastn, blastp, and blastx searches! Now, before you click the BLAST button, consider...Standard Protein BLAST (blastp) and blastx search to verify expected protein sequence in a plasmid. If you know...Addgene Blog: Plasmids 101: How to Verify Your Plasmid Plasmids 101: An Inside Look at NGS Plasmid Quality ...Search Tool (BLAST) provided by NCBI. BLAST helps us compare the sequencing results of the plasmids in our ...Choosing a BLAST program Of the five BLAST programs available, we primarily use Standard Nucleotide BLAST (blastn...differences in the blastn programs, check out the BLAST Help webpage. Optimizing blastx searches Once you...

... Redondo-Salvo, S., & de la Cruz, F. (2023). Plasmid classifications. Plasmid, 126, 102684. https://doi.org...toolbox. Natural plasmids, also called environmental plasmids, are all around us! A plasmid is a small piece...colicin-producing plasmids have been referred to as “Col plasmids” or “Col factors.” Virulence plasmids: microbial...Shareck et al., 2004). Plasmid categorization is complex Plasmid classification has shifted over the years...Old Solution but a New Problem Plasmids 101: What is a Plasmid? Plasmids 101: Transformation, Transduction...., 2001) Beneficial traits of natural plasmids Natural plasmids often convey a variety of functions that...functions of natural plasmids. Created with BioRender.com. Resistance (R) plasmids: microbial defense...

...Addgene blog Plasmids 101: Origin of Replication Plasmids 101: What is a Plasmid?  Plasmids 101: Transformation...host range in plasmids? Not all plasmids were created equally. Only a handful of plasmids have been identified...broad host-range IncQ plasmids. Plasmid, 62(2), 57–70. https://doi.org/10.1016/j.plasmid.2009.05.001  Meyer...define plasmid host range, identify a few of the features that confer broad host range in plasmids in nature...recombinant vectors.  A plasmid’s host range is determined by the different hosts that a plasmid can transfer to...influence the plasmid’s ability to establish and maintain itself within host cells.  The plasmid has multiple...if a plasmid has few restriction sites, it may be able to sneak past this checkpoint.  The plasmid is ...

...once in the plasmid backbone and another enzyme that cuts within the insert. If the plasmid contains the... PCR product if the plasmid contains the insert and no PCR product if the plasmid does not contain the...resources on the Addgene blog Get all the plasmid basics in our Plasmids 101 series Find more molecular biology...If you’re cloning a plasmid, you’ll need a way to find the needle in the haystack: the one perfect clone...clone that contains the plasmid you’re looking for out of the many cells that don’t. One way to begin ... how to use positive and negative selection in plasmid cloning.  But oftentimes, a selection strategy...enough on its own to help you find your desired plasmid. In many cases, you will need a screening strategy...

... successfully takes up plasmid DNA. Among those that do, some now have plasmids that contain your gene...including ccdB on its destination plasmids. In the destination plasmid, the ccdB gene is flanked by recombination...selections in plasmid cloning. Additional resources on the Addgene blog Read our Plasmids 101 blog posts...purify your gene of interest, get it into your plasmid backbone, and zap the mixture of DNA into cells...gene of interest, but others will uptake plasmid backbones that re-ligated back on themselves. Therefore...strategy needs to identify cells containing the plasmid construct you’re seeking. Fortunately, there are... example, if the DNA you’re inserting into the plasmid backbone contains a kanamycin resistance cassette...

... Bioblasts? Plasmagenes? In the 1940s and 50s, scientists were working to understand genetic cytoplasmic...how did plasmids get their name? In 1952, Joshua Lederberg set out to clarify the classification of these...these cytoplasmic inheritance factors. He proposed the catch-all term “plasmid” derived as a hybrid of ...tetracycline resistant plasmid, pSC101, and a newly developed kanamycin resistant plasmid, pSC102, with EcoRI...engineering, cloning, and plasmid tags, addtional content added to the "Plasmids that Glow" and "Viral Expression...Learn more about the history of plasmids and cloning: CSHL Meeting: Plasmids: History & Biology The Joshua...of "cytoplasm" and "id" (Latin for 'it'), as “a generic term for any extrachromsomal hereditary determinant...

...letter of a plasmid name and simply denotes that the object is a ‘plasmid’. “p” is for plasmid. pXXXXX-XXXX...information about plasmid features and tips you can use in the lab, check out more posts in our Plasmids 101 series...There are no universal rules for naming plasmids but here are some good guidelines to follow in order...people can quickly and easily identify what your plasmid contains and other important information. Tip:...Backbone name Include the empty backbone name in your plasmid name. This simple piece of information can often...important details. Once you know the backbone a plasmid is based on, you can usually derive: a) the bacterial...  Include information about the insert in your plasmid name. This is often a 3-6 letter representation...

...2017 and last updated Apr 6, 2021 by Will Arnold. Introducing NGS in 2017 for all incoming plasmids was a...every isolated plasmid sample and normalize each to fall within a 10 fold range. Plasmids that don't meet...A broader view of the whole plasmid To confirm the sequence of the plasmid, we examine three things: ...function of the plasmid.   Confirming the insert We usually confirm the insert through BLAST or by direct...expected and do not affect plasmid function, we will update information on the plasmid’s page. Sometimes we’re...’re missing a piece of the plasmid There are regions of some plasmids that are particularly difficult ...some plasmids will not result in one complete, circular assembly. Most of the time, these plasmids are ...

...the plasmid based on the predicted sizes and organization of different features within the plasmid. Restriction...if you don't have the full plasmid sequence. Once you have purified plasmid DNA, this method can be done...example plasmid on the right has a total size of 7.3kb, including a 1.2 kb insert. The plasmid was digested...possible plasmid conformations, with relaxed and nicked marked with asterisks (*). When the plasmid is digested... Congratulations, you have a plasmid expressing your gene of interest (YGOI) and are ready to dive into...functional experiments! Whether you’ve cloned the plasmid yourself or obtained it from a colleague down the...with the correct construct, and verify that the plasmid you received matches the expected sequence. Here...

... metabolic enzymes including biotin carboxylase and decarboxylases. These enzymes catalyze the transfer...biotinylating proteins with BirA. You can find plasmids that contain both AviTag and BirA at Addgene! ...

... on All Things Plasmid with Our Plasmids 101 Series Get Tips on Verifying Your Plasmid Resources on Addgene.org...Check out Our Plasmid Reference Pages Deposit Your Completed Plasmids Find the Perfect Plasmid for Your Research...recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid Bonus...recipient plasmid. Remember to insert your DNA in the correct orientation in the recipient plasmid by viewing... of DNA so that it can be easily cloned into a plasmid of interest. You can use similar processes to add... Interest (YGOI) for ligation into a recipient plasmid. Designing primers for PCR based cloning   ...this example). Assuming you are amplifying from plasmid DNA (rather than from genomic DNA or a cDNA library...

... (blue) light switch. Zhang’s team devised the plasmids now available in Addgene’s repository for use ...biological processes and disease states. While LITE plasmids have clear application to anyone with an interest...the new optogenetic tools in C. elegans and in Plasmodium. For those who do want to apply LITEs for the...

...of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  ...selectivity. With 10 plasmids available as a kit, plus 21 optional accessory plasmids, the collection targets...precise plant breeding. Find dicot prime editing plasmids here! Vu, T. V., Nguyen, N. T., Kim, J., Song... proteins for mammalian cell expression. These plasmids will be useful not only for future studies of ...proteins of interest too! Find CLIP, CLAP, and SNAP plasmids here! Smith, E.R., Turner, E.D., Abdelhamid, ...al., 2024. The authors have deposited AAV plasmids for Cre-dependent and -independent versions that...potential, ASAP5 is a great new option. Find ASAP5 plasmids here! Hao, Y. A., Lee, S., Roth, R. H., Natale...

...to find Gateway plasmids for your organisms of interest: Find Gateway cloning plasmids here! References...antibiotic resistance markers (indicated here by plasmid color), allowing you to easily select for the expression...desired, recombined construct. Read our recent Plasmids 101 Post on CcdB for more information. How to ...Method A: recombination of an attB-PCR product or plasmid with an attP donor vector. In this case, we would... to use the Snapgene program to design Gateway plasmids.    Figure 3: Method A to create an entry...entry clones can be used with a large variety of plasmids. Speed The Gateway system enables the generation.... Take a look at some of the Gateway Multisite plasmids available at Addgene, including the Frew Lab Multiple...