...Method C: Restriction cloning of a restriction enzyme fragment containing the DNA of interest and a attL-entry...scientists are no longer limited to traditional restriction enzyme cloning. Instead, you can choose a molecular...vectors, one can use Gateway to clone a gene of interest into a variety of expression systems. Keep reading...generates the entry clone containing the DNA of interest flanked by attL sites. As a byproduct of the reaction... a result, an expression clone with the DNA of interest flanked by attB sites is generated. As in the ...recombination, and it is exchanged with the gene of interest during the BP or LR reactions. Since the CcdB ...Figure 5: Method C to create an entry clone: Restriction cloning the inset into an attL-containing entry...

...the mutation of interest. Gel electrophoresis diagrams of the anticipated restriction analysis results... by Restriction Fragment Length Polymorphism (RFLP) analysis (right). Introduction of restriction site...conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to...the plasmid template (blue), and introduce a restriction site “A*” (in addition to the “A” site already...in the parent vector). Also shown are the “B” restriction sites, which flank the targeted region. Upon ...amplification, the template is removed by DpnI restriction digestion leaving behind only the mutagenized...primers to successfully anneal to the plasmid of interest during the PCR reaction. Ideally, your primers...

...institutional oversight is required. Restraint: mechanical restraint or anesthesia Location: tail vein or...you might after getting a vaccine. Restraint: mechanical restraint Location: thigh Gauge: 25–27 Volume... create xenograft models. Restraint: back scruffing or mechanical restraint Location: back or hind legs...Injection Route Restraint Location Gauge Volume Skill Intravenous Mechanical restrainer or anesthesia ...potential pain or discomfort of the mice, and restraint method. Many different substances can be injected... potential off-target pokes to nearby organs. Restraint: scruffing Location: peritoneal cavity Gauge: ..., like on the back. Depending on the site and restraint method, subcutaneous injections take some practice...

...selectively detecting the target-of-interest in your assay and sample-of-interest. The approaches available broadly...antibody can detect the antigen of interest in the protocol of interest, ideally at endogenously expressed...a candidate antibody can detect the antigen of interest in the given protocol. This requires use of one...ability of an antibody to detect the antigen of interest when expressed at endogenous levels (Laflamme ... a robust level, similar to that in samples of interest. If you are working with cells that are amenable...approach to antibody validation. If your protein of interest is essential to the cell’s function, it likely...on the specific antibody:target combination of interest (Figure 1). It can therefore be helpful to try...

... containing the protein of interest flowed through. The protein of interest binds to the metal ions, becoming... of interest. A high imidazole elution buffer disrupts the interaction of the protein of interest and ...the N- or C-terminus of a recombinant protein of interest. The tag was developed as a means to purify a ...proteins and other contaminants. The protein of interest is then eluted using a high imidazole buffer that...outcompetes the interaction between the protein of interest and the metal ions.      Fig. 1: A sample...sample containing a polyhistidine tagged protein of interest (purple) and several contaminating proteins (blue...through the Ni2+-containing resin. The protein of interest (purple) binds to the Ni2+ and is immobilized ...

...principle behind MXS-chaining is repeatable, restriction-enzyme based (chaining-based) cloning. The modules...Modules are assembled one at a time by standard restriction enzyme digestion followed by ligation. Upon ligation...the repeatability of the method. Note that the restriction sites of the MCS are only regenerated at the ... 3’ termini of the ligation product, and the restriction site between the modules is eliminated upon ligation... using these techniques are free of specific restriction enzyme recognition sites. Thus, chaining methods...Technology Pros Cons References Golden Gate Type II restriction enzymes Can assemble up to 10 modules in a single...reaction, can assemble large DNA sequences, no restriction enzymes needed Not optimal for joining sequences...

... am talking about making sure the audience is interested, educated and talking as a result of the content...where the questions are obvious, the answers uninteresting, the panel members all say the same thing and... a well-planned approach to making the event interesting and engaging. Long before the event... Choose... should be taking good notes and discovering interesting stories, riffs or opinions that each panelist...moderator should identify the funny, provocative or interesting stories that will make for a fun discussion. ...questions in a directed way to bring out these interesting stories and remind the panelists of the stories...spontaneity, but having a good handle on some interesting questions and topics in advance is key. Make ...

...scientific interests were or what I wanted to do with myself when I was 18. I just knew I was interested in trying...some perspective and advice to any undergraduate interested in getting a taste of the role of a scientist...begin. Here’s my advice: Reflect on your personal interests While it’s perfectly fine to be unsure, it helps...excited about the science allows you to ask more interesting questions and your excitement will in turn inspire...makes for an experience more tailored to personal interests. Talk to people, ask questions, and dig deep Talk...though, try to be conscious of who has your best interests at heart. A great piece of advice I’ve gotten ...who have taken career paths that you would be interested in can also help you clarify your goals and define...

...unwanted selection markers. Restriction modification systems Because restriction modification systems consist...toxin-antitoxin roles. Restriction modification systems are composed of restriction endonucleases and methyltransferases...preventing the cognate restriction enzyme from cleaving the DNA. A role for restriction endonucleases in plasmid... when the ccdB gene is replaced by the gene of interest. As a result, cells that retain the ccdB gene ...methyltransferases. Restriction endonucleases cleave DNA at or near specific nucleotide sequences. To ...Escherichia coli in 1995 when introduction of restriction-modification systems onto plasmids allowed cell...these plasmids as compared to those without the restriction modification systems. As with the other toxin-antitoxin...

...of fluorescent fusion protein with your gene of interest: Plasmid Color Expression Description pPROEX... mCerulean N1 Cyan Mammalian Express a gene of interest fused to the N-terminus of monomeric Cerulean ... mCerulean C1 Cyan Mammalian Express a gene of interest fused to the C-terminus of monomeric Cerulean ... tag SCFP3A Cyan Mammalian Express a gene of interest fused to the C-terminus of SCFP3A Amber N1 Yellow... Yellow Mammalian Express a gene of interest fused to the N-terminus of Amber Amber C1 Yellow Mammalian...Mammalian Express a gene of interest fused to the C-terminus of Amber mVenus N1 Yellow Mammalian Express...Express a gene of interest fused to the N-terminus of monomeric Venus mVenus C1 Yellow Mammalian Express ...

...fragments, but uses site specific restriction sites (Type IIS restriction endonucleases) to cleave DNA outside...which have provided appealing alternatives to restriction enzyme cloning. These newer technologies have...They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on....that does not require the presence of specific restriction sites (or lack thereof) nor a serious time commitment..., imagine that you want to insert your gene of interest into a vector with a large tag at the N-terminus.... If you inserted these two pieces of DNA by restriction enzyme cloning, you would have to do it in two...Gibson assembly, you can insert both the gene of interest and the tag sequences into the vector in one step...

...relies on Type IIS restriction enzymes, first discovered in 1996. Type IIS restriction enzymes are unique...If your genes of interest or destination vector contain multiple internal restriction sites that may not...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method has...unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, ... The cloning scheme is as follows: the gene of interest is designed with Type IIS sites (such as BsaI ... ligation favors formation of the construct of interest. Although efficiency may decrease with an increased...to as MoClo, this strategy uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble...

...protein of interest The first step in analyzing the localization of your protein of interest is to make...disease, researchers often isolate proteins of interest and examine them using biochemical methods thus...healthy and diseased states can also provide interesting insights into disease mechanisms and protein ...available for microscopy, by cloning your protein of interest into a vector encoding a fluorescent tag. Once...fusion, it is possible to track your protein of interest from its production site to its final destination...traffics the fluorescent protein to your organelle of interest. Click on the gene names in the figure below to...plasmids for labeling your subcellular structure of interest in the Allen Institute for Cell Science Plasmid...

...screen using PCR and a restriction enzyme digest.   Fig. 3: Restriction enzyme screening for resistivity...edits.   Interested in this method, but your edit doesn’t affect any endogenous restriction enzyme sites...screen hundreds of clones looking for your edit of interest. For all of the screening strategies outlined ...introduces a frameshift in the coding region of interest will get the job done. In other words, any insertion...First, use PCR to amplify the target region of interest. Then Sanger sequence the PCR product of both ... 50% are -/+, and 25% are +/+ for your gene of interest. Thus, to find a null clone, you should screen...of your experiment for a screening strategy. Restriction enzyme screening is sensitive to single base ...

...express your gene of interest either constitutively or inducibly. If your gene of interest is toxic or needs...If you are interested in using lentiviral vectors to introduce your favourite gene into your favourite...and the transfer plasmid encoding the insert of interest as well as sequences necessary to encapsulate ...system was efficient at transferring the gene of interest to the appropriate cells, it was obvious that ...The ability to infect non-dividing cells is not restricted to in vitro cell culture as lentivirus-derived... optimum concentration to infect your cells of interest.   Advantages Disadvantages Can carry large...transfer plasmid is the vector you clone your gene of interest into and is an important feature for your experiment...

...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...that sequence modifications can only be made at restriction enzyme cut sites. The lambda red system is an... of DNA within E. coli and is independent of restriction sites. The lambda red system is derived from ...means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic changes occur...sections, key steps that differ from traditional restriction enzyme cloning will be explained in greater detail...Red recombineering system to replace a gene of interest with an antibiotic resistance cassette.   ...using primers that amplify the DNA sequence of interest and flank it with 50 base pairs of homology to...

...with Type IIS restriction enzymes and standardized primers to clone your gene of interest into both sequencing...protein of interest. The plasmids used to generate these fusions all use the same restriction enzymes for... pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance... they are unlikely to be found in your gene of interest) and prevent incorporation of extensive sequences...kit will allow you to quickly study your gene of interest in a variety of model systems, thereby opening...downstream of which you can clone your gene of interest. Translation of the standardized leader peptide...secondary structures in the mRNA encoding your gene of interest from affecting its translation initiation and ...

...finding new factors in your biological system of interest, the so-called “fishing expedition.” Your first... process in which only cells with the trait of interest will survive in the selected growth conditions...hinder, or have no effect on the process you are interested in. What does this actually look like? For a...selected. The method you choose to assess traits of interest is highly specific to your scientific question...CRISPR, but instead of knocking out the genes of interest (or the whole genome), they knock down gene expression...RNAi libraries are especially useful if you are interested in studying genes whose knockout would be lethal...apply enough pressure to select for variants of interest, though. A selection can take the form of varied...

...targeting or cloned in-frame to the protein of interest for plasmid-based expression systems. Degron tags...is situated between the degron and the gene of interest and the degron domain is terminal. Between the...elements (protease and degron) and the protein of interest, there is also a protease recognition site. In...the presence of the degron, and the protein of interest is untagged and functions per the usual. In the...FKBP12F36V. By fusing FKPB12F36V to your protein of interest, it can be degraded by this selective degrader...fusion protein. This will target the protein of interest to the proteasome for degradation. Like several...dimerization of an FKBP12F36V fused target protein-of-interest and the CRBN E3 ligase complex leading to ubiquitination...

... on versatile Type IIS restriction enzymes. These cut outside their restriction sites allowing you to ... But...how? Cloning is no longer limited to restriction enzymes - although many labs still love using...extensively described in other articles; so if you are interested in learning more about any one of them, please...your E. coli do the work for you. Traditional restriction enzyme and ligase based cloning is a relatively...The ligase cost is comparable to that of the restriction enzymes and overall you’re looking at a price...of a few dollars per reaction for traditional restriction cloning. Sequence and Ligation Independent Cloning...appropriate inserts by PCR, there is no need for restriction digests, gel purifications, or ligations. The...