...cloning plasmids, expression plasmids, gene knock-down plasmids, reporter plasmids, viral plasmids, and ..., modify, or construct a plasmid. A plasmid is a small circular piece of DNA found in bacterial cells,... of scientists and plasmid lovers - serves as an introduction to plasmids. Plasmids 101 will provide you...  What is a plasmid? At their most basic level, plasmids are small circular pieces of DNA that replicate...that make up a plasmid and why each is important. Our “Plasmids 101” series designed to educate all levels...contrast, plasmids utilized in the lab are usually artificial and designed to introduce foreign DNA into another...cloning site. The ease of modifying plasmids and the ability of plasmids to self-replicate within a cell ...

...levels of protein, not plasmid DNA. If you need large amounts of DNA from a plasmid in a protein expression...to an improved plasmid DNA yield. Culture conditions play a critical role in plasmid DNA yield. Different...co-purify with your plasmid DNA and lead to degradation of your prep. Many plasmid prep kits recommend...more cells = more plasmid. Plasmids can vary in copy number, the number of plasmid copies supported within...sub-culture, and prep with a DNA prep kit or your lab's favorite in-house protocol. DNA yields for this procedure...excess of 100 ng/ul, more than enough DNA to verify your plasmid via sequencing or restriction digest....where your plasmid yield is sub-optimal? If you are consistently getting sub-optimal plasmid yields from...

...elute and store your plasmid DNA Learn about the different grades of plasmid DNA preps Resources on Addgene.org...signs of DNA in your final tube after purification. How do you know if you actually have DNA in your tube...of the DNA sample.  These absorbance measures give you an idea of the concentration of your DNA prep and...sensitivity at low concentrations of DNA and it can’t distinguish between DNA and RNA. See our protocol about...UV absorbance to quantify DNA Fluorescence dyes Another way to quantify DNA would be to use fluorescent...way to quantify DNA, you can use the agarose gel method to not only find out how much DNA you have, but ...fluorometer to quantify DNA this way. First, start by pouring your gel containing a DNA intercalating dye (...

... ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure 1: Transformation...process of inserting exogenous DNA in a microorganism. The amount of plasmid DNA that is necessary for bacterial... into a plasmid backbone. Transfection grade DNA Figure 3: Need to introduce plasmids into mammalian...utilize your plasmid DNA, so that you can decide what will be the optimal type of plasmid preparation ...Not all plasmid preps are the same. Before purifying a plasmid from a bacterial culture, it is important... of DNA you need, and at which level of purity. Based on these premises we can classify a plasmid preparation...transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation is the...

...fragmenting plasmid DNA, PCR product, or chromosome. One of the main causes of degradation during plasmid DNA ...want to use the plasmid DNA can help you choose the best way to store and elute the DNA. References Murakami... to purify DNA without a kit Read our blog posts about plasmid cloning Browse our Plasmids 101 blog posts...experiments you can effectively use the DNA. pH and DNases affect DNA stability Figure 1: So many choices...responsible for the sugar backbone of the DNA helix or even changes the DNA sequence through a mechanism called...adenine or guanine) from the DNA. These apurinic sites if in double stranded DNA are repaired by base excision...degradation and which experiments you can use your plasmid.  DNA elution choices: TE, Tris buffer, or water ...

... you don't have the full plasmid sequence. Once you have purified plasmid DNA, this method can be done...Always run control uncut DNA to ensure your enzymes are working. When uncut plasmid DNA is isolated and run...the plasmid based on the predicted sizes and organization of different features within the plasmid. Restriction...steps: 1) incubating your DNA with restriction enzymes which cleave the DNA molecules at specific sites...example plasmid on the right has a total size of 7.3kb, including a 1.2 kb insert. The plasmid was digested... as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed and ... pouring it. EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light. Don...

...and genomic DNA to precipitate. Plasmid DNA is free in solution. Proteins and genomic DNA are removed ...agarose gel slices Product: plasmids or DNA fragments The plasmid purification and DNA gel extraction method...modifications depending on the type of DNA you’re purifying (plasmid, genomic, or DNA fragments from agarose).    ...alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free...solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA, the glass particles... Read our Plasmids 101 blog series Find other plasmid protocols and tips Browse our plasmid cloning blog...column, stop to consider some ways to purify DNA without a kit. DNA purification kits have advantages: they...

...weight genomic DNA; filter the remaining plasmid DNA through a column that binds the plasmid DNA and lets other... your insert (or donor plasmid) and plasmid backbone. Because you lose some DNA during the gel purification...restriction enzymes to cut open a plasmid (backbone) and insert a linear fragment of DNA (insert) that has been ...restriction enzymes. An enzyme, DNA ligase, then covalently binds the plasmid to the new fragment thereby ...restriction cloning process. Both the plasmid (blue, backbone) and the DNA sequence of interest (green, insert...insert and 1μg of plasmid backbone. It is also critical that as much of the backbone plasmid as possible be... of the backbone plasmid (see below). You should treat your digested backbone plasmid with a phosphatase...

...general, these plasmids tend to be low copy number. Conversely, plasmids that can initiate DNA replication...growth against sustainability. Plasmid DNA replication and regulation Plasmids must also strike a balance:...nearby AT-rich regions of DNA. In a test tube, the DNA helix can be separated by DnaA alone, as long as there...replication of the host cell's chromosomal DNA.  Plasmids are said to be under stringent control when...replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...at an example plasmid, let's review how E. coli normally replicate their chromosomal DNA. Chromosomal ... ori. In the elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication fork....

... successfully takes up plasmid DNA. Among those that do, some now have plasmids that contain your gene...of interest, get it into your plasmid backbone, and zap the mixture of DNA into cells. Unfortunately, not...antibiotic. For example, if the DNA you’re inserting into the plasmid backbone contains a kanamycin resistance...been transformed with the plasmid backbone (with or without your inserted DNA) and further screening will...gene is found on the original plasmid and either the insertion of a DNA fragment within the gene or loss...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...

... you are amplifying from plasmid DNA (rather than from genomic DNA or a cDNA library), roughly 18-21bp... 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...and NotI to ligate our cDNA into the recipient plasmid. Remember to insert your DNA in the correct orientation... piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it...recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid Bonus...essential that you verify your final plasmid by sanger sequencing. DNA replication by PCR has error rates...

...fragments of DNA to create a synthetically designed DNA sequence. There are multiple methods of DNA assembly... scarless plasmid inserts.  Want to Learn More about DNA Assembly? Check Out Our Plasmids 101 eBook!...New England Biolabs. What is DNA assembly? In the context of cloning, DNA assembly refers to a method ... NEBuilder® HiFi DNA Assembly. Gibson Assembly allows the production of scarless DNA constructs using ...shuffling of DNA regions. Golden Gate Assembly also allows the creation of standardized DNA constructs,...homology) cut from plasmids via restriction enzyme digestion, and used directly in DNA assembly will still...as easy as obtaining a plasmid with the gene of interest (search Addgene plasmids by gene here), designing...

...adjacent lysed bacteria and can include plasmid DNA or fragmented DNA released into the environment. Various...don't exactly "eat" plasmids as depicted in this comic, when a bacterium uptakes a plasmid, the genes on the... the plasmid can give them different phenotypes. For example, this plasmid encodes a green fluorescent...bacterial DNA can be accidentally packaged into the newly created phages. Transfer of this DNA to another...Transferred DNA once inside the infected bacterium can either exist as transient extrachromosomal DNA, like ...commonly use phagemids, a DNA cloning vector that contains both bacteriophage and plasmid properties. The phagemids... Read all of our Plasmids 101 blog posts Check out all of our blog posts on plasmid cloning Resources...

...to Elute and Store Your Plasmid DNA? by Paolo Colombi Should I store my plasmid in TE, Tris, or water?... a subset of these tools for plasmid mapping, DNA/protein databases, DNA sequence manipulation, and primer...new cloning grade DNA service. We've had 11 blog posts on COVID-19 featuring plasmid tools from depositors...launched the cloning grade DNA service. This service features 210 plasmids: empty backbones, fluorescent... tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning grade DNA to you, we'd save ... development virtually. 6. Quantifying DNA? Here are Five DNA Quantification Methods to Consider by ... dominates DNA quantification discussions, but what about the other methods to quantify DNA? Here we cover...

...Unmethylated DNA not degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated DNA not degraded...cytosine DNA Allows for uptake of foreign (methylated) DNA recA, recA1, recA13 Mutation in a DNA-dependent...recombination and general DNA repair Reduces plasmid recombination, increases plasmid stability recBCD Exonuclease... routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many mutations that make...improvements such as those that increase plasmid yield and/or DNA quality. Table 1 below outlines a few ... Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying...ClaI or XbaI) dcm DNA cytosine methylase mutation (CCWGG) Preparing unmethylated DNA, important when trying...

...using plasmid tools. Details about the cloning grade DNA beta project We will ship you  2 µg of DNA in ... after ordering a plasmid you didn't have to grow the bacteria and prep the plasmid before you begin your...grade DNA service and is ready to use out of the box. We estimate that providing cloning grade DNA could...discovery.” Here are the details. Plasmids offered as cloning grade DNA As we begin testing out this service...with 210 plasmids from the collection. You can find the full list here. Most of these plasmids are empty...which plasmids you’d like to see as part of the service in the future using the Cloning Grade DNA Suggestion...blog posts from our Plasmids 101 blog series Learn about the different grades of DNA  Read about the various...

...from foreign DNA. The other component of these systems, methyltransferases, methylate DNA at particular... specific DNA sequences and companion endonucleases that recognizes and cleave the same DNA sequence if...stretches of DNA: Dam methylase adds a methyl group to the adenine of GATC stretches of DNA Dcm methylase...desired mutation into your plasmid of interest by PCR generates mutated plasmids with no methylation (there...Mutagenesis to Mutate Your Plasmids Resources on Addgene.org Check Out Our Plasmid Protocols Boost Your Molecular...Or considered why DpnI will degrade your template DNA from a PCR reaction but not the newly synthesized...the same--methylation! Read on to learn about how DNA methylation may affect your restriction digests. ...

...underrepresented plasmids (plasmids that were lost or “dropped out”) and overrepresented plasmids (plasmids that ...In addition to single plasmids, Addgene also distributes pooled plasmid libraries containing hundreds...transformed with the library DNA, they grow and replicate, and then their DNA is harvested, much like one...one would prep an individual plasmid. However, lots of library DNA is needed for screening experiments...so the scale of the DNA prep is larger. And since the library contains many plasmids, care must be taken...diversity. If the various plasmids in the library are differently sized, smaller plasmids may be amplified preferentially...pelleted for DNA extraction. Prepare library product for next-generation sequencing Once the DNA has been ...

...called DNA transposons. DNA transposons are commonly used as tools in the lab, so we’ll focus on DNA transposons...move antibiotic resistance genes between plasmids or from plasmids to the bacteria’s genome. In humans, if...Transposon: The transposon is the DNA sequence that moves around. This DNA sequence encodes the proteins ...BioRender.com.   Class II TEs: DNA transposons Class II TEs are also known as DNA transposons, since they do...break, and excises the transposon from the donor DNA. A DNA footprint is left behind. When the transposon-transposase...not leave behind a DNA footprint sequence after excision. piggyBac can mobilize DNA over 100 kB in size...transposase.  piggyBac plasmids    Tol2  Tol2 was the first reported active DNA transposon in vertebrates...

...Commercial DNA Ladder Penn State Ladder: Homemade Plasmid Prep Penn State Ladder: Commercial Plasmid Prep Kit...Two plasmids that can be used to make inexpensive 100 bp or 1 kb DNA molecular weight ladders were recently...developed the plasmids, pPSU1 and pPSU2. When restriction digested with PstI or EcoRV, these plasmids generate...Verifying Your Plasmid Table 1: Comparing Typical Comericial Ladders with the PSU Ladders DNA Lader Commercial...interested in making your own DNA ladder? You can find the Tan Lab’s DNA ladder prep protocol here and...Johnston, J. L., & Tan, S. (2017). The pPSU Plasmids for Generating DNA Molecular Weight Markers. Scientific ...generate 100 bp or 1 kb DNA ladders, respectively. Unlike many commercially available ladders, the 100 bp ladder...