... my poor, poor legs. Running for FOP, running for AJ Runners for #Running4Rare are paired with patient... I learned there was a Genzyme-based running team called Running for Rare Disease that runs on behalf ...mall. I took action, joined a gym, and started running on a treadmill. After being with the company for...parents, Kristi and Rico, had to keep him from running too much as any jarring of his body could initiate...raising funds and awareness of FOP. Please consider running with us or making a donation via the link below...the emotion HOPE; a fitting superpower for those running on behalf of rare disease sufferers like AJ. As...development for First-In-Man studies. Kurt has been running on behalf of AJ Gonzales, a young boy suffering...

...these include, TA cloning, ligation independent cloning, TOPO cloning, one step cloning, and overlap extension...Standard Restriction Cloning and Fast Cloning Requirement Restriction Cloning Fast Cloning Choose Restriction...restriction cloning is still routinely performed by many labs, a variety of other cloning techniques with...excessive time requirements. FastCloning is a simpler yet reliable cloning technique that was developed... Transformation + + The FastCloning process in brief In FastCloning, you PCR both the vector and ...and ligated in vivo. Detailed FastCloning procedure To start the FastCloning process, the insert and vector...the multi-cloning site, and the reverse primer is located in the 5’ end of the multi-cloning site. The...

...a look at a few screening methods. Blue-white screen A widely used method of screening is the blue-white...biology, cloning a gene into your plasmid is often a numbers game. By using a variety of screening and selection...If you’re cloning a plasmid, you’ll need a way to find the needle in the haystack: the one perfect clone...use positive and negative selection in plasmid cloning.  But oftentimes, a selection strategy is not ...desired plasmid. In many cases, you will need a screening strategy as well. In a screen, you aren’t killing...selection, then screen Why add a screen to your cloning strategy? A screen will help you more easily identify...resistance marker in the backbone. A selection on agar containing ampicillin will yield bacteria that have taken...

...directional cloning   The principle behind MXS-chaining is repeatable, restriction-enzyme based (chaining-based...between various cloning methods are listed in Table 2. For example,  chaining-based cloning methods require...various chaining-based cloning methods are listed in Table 3. The goal of high throughput cloning is to ....org. Table 3: Chaining Based Cloning Methods Method Restriction Enzymes Chaining Direction MXS-Chaining...a useful application of high-throughput cloning. MXS Chaining is one such technique and has been used ...Neveu, 2015). Origin and purpose of MXS chaining MXS-chaining was designed to create plasmids for fluorescence... generated MCS. MXS-chaining also has the advantage of being directional, meaning that (in the previous...

...plasmid. Designing primers for PCR based cloning     The basic PCR primers for molecular cloning consist...outlined in our restriction cloning post with a few quirks specific to the PCR cloning process - please check...our restriction cloning post. However, even more than with standard restriction cloning, it is essential...In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time...Restriction Site: Your chosen restriction site for cloning (usually 6-8bp) Hybridization Sequence: The region...your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid...these are used for primer design: Because we are cloning an ORF, we want to clone from the start codon (...

... bacterial in vivo cloning is now able to come of age. The in vivo assembly cloning protocol In Vivo Assembly... to drive assembly. Combining multiple modifications A key feature of IVA cloning is the ability to combine...assembled using IVA cloning, although the efficiency of assembly decreases as the cloning complexity increases...Watson, and Ingo H. Greger. "IVA cloning: a single-tube universal cloning system exploiting bacterial in...before choosing a cloning method Check out all of our blog posts on plasmid cloning Resources on Addgene.org...the MRC Laboratory of Molecular Biology. Plasmid cloning is an essential part of any molecular biology project... experimental process. The majority of current cloning techniques involve the assembly of a circular plasmid...

...strangers to cloning. If you’re reading this, then chances are you have been in the cloning trenches yourself...yourself. Modular cloning, or MoClo, is a systematic cloning strategy designed by synthetic biologists...experiments it enables. What is modular cloning? Modular cloning, or MoClo, is an organization scheme for... MoClo reactions use Golden Gate cloning, not all Golden Gate cloning is MoClo. That’s because MoClo specifically...biologists to perform large scale, one-tube cloning reactions. In this blog, we’ll cover the basics of what MoClo...hierarchy. MoClo originally developed from Golden Gate Cloning, in which a type IIS restriction enzyme and a ligase...schema described above, and not any particular cloning technique. Though rarer, you can find MoClo that...

...attL-entry-TOPO vector. TOPO cloning adds short end(s) to facilitate cloning into an attL-containing entry vector.   ...TOPO cloning the insert into an attL-containing entry vector.   Method C: Restriction cloning of a...facing a cloning project, scientists are no longer limited to traditional restriction enzyme cloning. Instead...Gateway cloning method and its advantages. An introduction to Gateway technology The Gateway cloning method...PCR product with an attP-containing donor vector.   Method B: TOPO-cloning of the desired insert into... is inserted in a multiple cloning site (MCS) of an attL-containing entry vector.   Figure 5: ...create an entry clone: Restriction cloning the inset into an attL-containing entry vector.   Pro Tip: ...

...Topoisomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes...TOPO-TA) cloning; however, the TOPO cloning technique has also be adapted for blunt end cloning. So how...researcher to perform blue-white screening after transformation - self joining of the vector ends results in... of TOPO cloning happens. Basic procedure Let’s break down the steps needed for TOPO cloning: 1. Create...on the basic ability of complementary basepairs adenine (A) and thymine (T) to hybridize and form hydrogen...how does TOPO cloning work? As illustrated in the Figure below, the “A” overhang on the blue PCR product... endonuclease and as a ligase by cleaving and rejoining supercoiled DNA ends to facilitate replication...

...Transformation containing the ligation mixture with backbone alone; 2. Transformation containing ligation mixture...When cloning by restriction digest and ligation, you use restriction enzymes to cut open a plasmid (backbone... digests.   Overview of the restriction cloning process. Both the plasmid (blue, backbone) and ...the insert and backbone covalent.  Before beginning the restriction digest and ligation process, you... proper orientation. For instance, if you were cloning a gene into an expression vector, you would want...downstream of the promoter as part of a multiple cloning site (MCS). Having multiple sites allows you to...it via their different migration speeds; after running the gel for some duration of time, these differently...

...dried out and died. The alternative to cloning rings, dilution cloning into 96-well plates, is extremely time... to the expense of dilution cloning. Taking a page from bacterial cloning, we reasoned that we should...Tips for using the agarose cloning method Test your agarose temperature. Cloning cells using an agarose overlay... laboratory became frustrated with using glass cloning rings to isolate colonies of antibiotic-resistant...time required to grease, place and fill a dozen cloning rings, the remainder of the colonies on the plate...thin layer of molecular biology-grade agarose containing physiological concentrations of salts and glucose...glucose. If we circled well-separated clones containing more than 50 cells (visible to the naked eye) with...

...Advantages of Golden Gate cloning Golden Gate cloning is one of the easiest cloning methods in terms of hands-on...enzyme-based cloning methods. Each method has its own pluses and minuses, but Golden Gate cloning has been... and easy-to-use system to your cloning efforts. Golden Gate cloning technology relies on Type IIS restriction...construct, allowing for precise, scarless cloning. The cloning scheme is as follows: the gene of interest... leveraged the power of Golden Gate cloning into a modular cloning strategy. Sometimes referred to as ...target sequence into a Cas9-containing plasmid such as pX330. This cloning strategy not only makes it ...easy.  Disadvantages of Golden Gate cloning Golden Gate cloning is not 100% sequence-independent: to ...

...and ease of generating gRNA-containing plasmids. CRISPR genome wide-screening experiments commonly use lentivirus...means to carry out so-called “forward genetic screening experiments”, it is certainly the most robust....repaired through error-prone non-homologous end joining (NHEJ), usually results in a loss-of-function mutation...genome-wide screens? The goal of a genome-wide screening experiment is to generate and screen a population...CRISPR can be readily scaled up for genome-wide screening due to the broad range of potential target sequences...consist of a heterogeneous population of gRNA-containing lentiviral transfer vectors, each targeting a...knock out, activate or repress target genes by combining a gRNA library with the aforementioned derivatives...

...insert (blue-white screening). Other types of screening methods Although blue-white screening is probably the...talk about an even more specific method of screening your cloning reaction. Being able to select for colonies... that! (To read about other screening strategies, check out our screening overview blog post.) *Note:...Blue White Screening Available at Addgene Are: pUC18 and pUC19 Let’s begin at the beginning. The well-...plasmid, creating an α-complementation cloning vector. When a cloning reaction goes to plan and your DNA ...Blue-white screening only indicates the presence of AN insert, not necessarily YOUR insert. Any cloning artifact... few common vectors. Blue-white screening is just that – a screening process. It does not select only...

... many more suggestions on planning for future success by finding and joining your dream lab, watch the... joining your dream lab...

...the steps in the cloning process, there are also many ways to troubleshoot the cloning experiment. Here...a different cloning strategy. Once you've identified potential clones after the screening, sequence the... DNA Custom Cloning Service since 2009. He has  developed a proprietary, low cost cloning protocol that... contributed by Oskar Laur, head of the custom cloning core at Emory University, and Paolo Colombi, a ...a product development scientist at Addgene. Cloning can be quite an arduous process. The PCR could fail...Here are some tips that will help you with your cloning project, and hopefully obtain your coveted plasmid...diving into the experimental work, spend some time outlining the construction of the plasmid and all the steps...

...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene... where you might need some help. Today: Career Planning Resources. The key to ensuring success during ...any formal or mandatory professional development training. Further, even for those students and researchers... page, where you’re likely to find current job openings. A good approach is to do this first for companies...way, once you’ve identified an appropriate job opening, you can send your contact an email with the relevant... graduate student or post-doc in a traditional training program, and you hope to make a career in academia...own Joanne Kamens, a strong advocate for career planning, has written a great blog on choosing good mentors...

...in a sample. Digital PCR assays for mutation screening Now that we have introduced how digital PCR works...indel mutations resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome ...the bi-allelic mutations typically desired when screening clones for complete functional knockout of your...interested in harnessing the power of digital PCR screening to validate your genome edits, this paper provides...Remember that although digital PCR is a great screening tool, it still requires target DNA sequencing ...PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells. PLoS ONE 11, e0153901–...

...your rate of interactions when screening! Tips for two-hybrid screening Minimizing false positives: Run... these discoveries to create a new system for screening protein-protein interactions. In later years, ...reporter gene cloned downstream of a promoter containing the target sequence of the DBD. The DBD-POI fusion...protein interact and enable intein splicing (5). Screening with yeast two hybrid systems While initial studies...plasmid transformants into a diploid strain for screening), or arrayed on a 96 well plate where each well...a defined prey fusion.  The former allows for screening greater numbers, but the latter eliminates the...the step of identifying prey proteins after screening. It is also possible to screen libraries of bait ...

...should acquire during their PhD and postdoctoral training. Addgene executive director Joanne Kamens has ...scientific society in your field. My leadership training began with the NPA where I gained invaluable experience...function without long distance communication and learning to work with people despite the distance improved...leadership institute’ weekends would entail intense training to develop our leadership and management skills...junior leaders what I had experienced and learnt. Opening doors to further opportunities  Having gained leadership... students to think about their career options, gaining transferable skills such as leadership skills can...