... as an example of LIC experimental design. Search Addgene's collection of empty LIC cloning vectors Protocol...plasmid together through the transformation/replication process. LIC employs long overhangs to form a stable...product is then repaired during the replication cycle. Empty vectors for LIC typically employ type II restriction...Protocols Overview Ligation Independent Cloning (LIC) obviates the need for the time-consuming ligation...in the example shown here. The manufacturer of an LIC-specific vector will provide the homologous sequence...Protocol Step 1: Design Your Primers Primer design for LIC is often as simple as using the backbone manufacturer's... protocol . Search Addgene's collection of empty LIC cloning vectors...

...26094 pET28a-LIC EF442785 Hexahistidine tag with Thrombin cleavage, KanR 26101 pET28GST-LIC EF456739 GST-tag...cleavage, KanR 26098 pCW-LIC EF460848 No tag, double ptac promoter, AmpR 26099 pFBOH-LIC EF456740 Hexahistidine...AmpR 26108 pFB-LIC-Bse EF199842 Hexahistidine tag with TEV cleavage, AmpR 26095 pFHMSP-LIC-N N/A Expression... Bac-to-Bac Expression System, AmpR 26100 pFHMSP-LIC-C N/A Expression vector for secreted proteins with...proteins of medical relevance, and place them in the public domain without restriction. Many SGC plasmids from...cart, then complete the checkout process online. Click here for additional ordering information....

... a single reaction Ligation Independent Cloning (LIC) Scarless cloning with Type II restriction enzymes...protocols that you can use for a wide range of applications, with videos for select protocols in the right-hand...protocols are the building blocks for many more complicated procedures. Plasmid Cloning Protocols for constructing...preparations. Antibodies Protocols for common antibody applications. Intro to the Lab Bench Name Description (Link...

... promoter pFastBac LIC and pFastBac Dual LIC - Vectors from the Gradia lab pFB-LIC-Bse - Insect expression...cells His-tagged versions of pFastBac LIC vectors and pFastBac Dual LIC vectors for insect expression Fluorescent...mammalian expression, Ligation Independent Cloning (LIC) pcDNA3.1-mCherry - Expresses mCherry tag in mammalian...bacterial expression, Ligation Independent Cloning (LIC) Also see our Bacterial Expression collection page...Trx (thioredoxin) Protein purification pNH-TrxT - LIC cloning in bacteria with N-terminal His-TrxA-TEV ...plasmids containing only the elements needed for replication in the host cell and are designed for a user ...our dedicated Cre-lox Plasmids page pBF3060 - Metabolic engineering in S. cerevisiae TALENs Gene targeting...

...the origin of replication/replicon. The replicon is comprised of the origin of replication (ori) and all...origins of replication There are lots of origins of replication out there so, for simplicity’s sake, we'... cells. The replicons of plasmids are generally different from the those used to replicate the host's ...gene for replication (reference). **F1 is a phage-derived ori that allows for the replication and packaging...control elements. The ori is the place where DNA replication begins, enabling a plasmid to reproduce itself...readily at lower temperatures and allows the replication machinery room to come in and get busy making...derivatives), pSC101, R6K, and 15A. Not all origins of replication are created equal. Some will produce many plasmid...

...previous nosy question! The Application Process Broadly speaking, the PhD application process tends to go something...steps in the application process and usually occurs in the few months leading up to applications opening. ...program. Most applications will recommend that you name two professors in your application that you intend...to continue the application process through a campus visit, a weekend where applications can get to know...strengthen your application and help you decide if a program is a good fit for you. The application process looks...know that this process only became even more complicated with COVID-19 and standards for hybrid, virtual...out above, there are many places where the PhD application cycle becomes much more complex. “How do I reach...

...plasmids rely on their host cell's replication machinery in order to replicate—but not always to the same extent...described in our previous Origin of Replication post, DNA replication is initiated at the ori and may or...consider the replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...review how E. coli normally replicate their chromosomal DNA. Chromosomal replication in bacteria is carefully...entire round of replication. Initiation is the primary control point of DNA replication in bacteria, so..., it recruits helicases to continue unwinding the strands. This creates two replication forks that move...chromosomal DNA ensure that the helicases are released from the DNA once the replication forks reach each other...

...resistance cassette, and the R6K origin of replication to allow replication only in hosts carrying the bacteriophage... an allelic exchange experiment? Find the pTOX plasmids here! Continued development of new allelic exchange...modification. This technique is frequently called allelic exchange. Here, a mutated allele introduced from...to off-target mutations. Ultimately, successful allelic exchange requires two sequential homologous recombination...counterselection vector is the pDS132 sacB-based allelic exchange vector. This vector contains sacB, an ...recipient of interest. Because pDS132 cannot be replicated in the recipient, the only antibiotic resistant...resistant colonies isolated are those where the allelic exchange vector has undergone single crossover and ...

... science policy, there is often discussion of “big P” and “little p” policy.  “Big P” policy is what you...about where science policy happens.  When do you do science policy? Many science policy training programs... is science policy?”  I have been asked this many times throughout my short science policy career and ...importantly, how, of a science policy career.  Who does science policy? I’ve intentionally started with... many science policy professionals that entered the field from a more traditional policy background, learning...to start. What is science policy? THE IMPOSSIBLE QUESTION. Science policy is the work you do to creatively... the National Science Policy Network to connect with early career science policy groups, attend webinars...

...disagreements. In fact, conflict resolution isn’t just about resolving conflicts between other people, ...how to sell them. Types of Conflict Before explaining my approach to conflict resolution, it’s important...there are different types of conflict. Most people perceive any conflict as inherently bad, and in many...harmful conflict early, because time frequently does not heal these types of wounds. But conflict can also...-that conflict you are resolving--will make you a better scientist. And some amount of conflict is actually...avoid all conflict can fall into “groupthink” and fail to evolve. Recognizing when “bad” conflict can be ...be spun into “good” conflict is a skill in and of itself. Conflict can also be categorized as either “personal...

...relies on heat resistant DNA polymerase from the thermophilic bacterium, Thermos aquaticus (Taq). Taq polymerase...extension is repeated 25-35 times to exponentially replicate the target DNA of interest. This entire reaction...have been developed for different scientific applications. These PCR methods all use the same basic PCR...the quantity of DNA after a PCR reaction as the amplicon doubles every reaction cycle. However, it is common...goal is to amplify a specific target of DNA. This amplicon can be used in molecular cloning to create a plasmid...quantified by qPCR. qPCR is used in a variety of applications including gene expression profiling, studying...concentration, unlike qPCR where results can vary across replicates. ddPCR can be used to quantify DNA sequences ...

...to breathe!)   Public speaking as a transferable skill The worst thing about doing public speaking as a...Public speaking is not just one of the most common phobias–it’s a key part of being a scientist and an...turned science communicator, I’ve done a lot of public speaking, and I'm happy to share what I've learned...would let me accomplish my goal.   As a scientist, public speaking isn't a performance art. You’re (usually...goal. Yay!   You’re only human When it comes to public speaking, Murphy’s law definitely applies: what...who do are very likely to be sympathetic because public speaking is hard. Aside from the diving board charades... and I've had multiple jobs where all I did was public speaking. If you're also nervous, try taking a ...

...their use in other clinical applications. In the last decade, viral applications in cancer treatment and ...s unpack the myriad number of emerging viral applications and give you some recommendations for putting...putting viruses to work. Viral applications Viruses work by infecting a host cell (the target of viral infection...expressed. It’s important to note that for all the applications we discuss, there is more than one type of virus...retrovirus, AAV, etc.). When you’ve decided on an application, our Viruses as Biological Tools blog can help...viruses and pick one for your experiment. Research applications Gene expression control Researchers are always...becoming increasingly popular tools with many applications. These libraries include sgRNAs targeting the...

...GST tag and TEV cleavage site. Includes sites for LIC cloning, and a "stuffer" fragment that includes the...containing a variety of tags can be found here . pBAD LIC Series (8-Series) Various Plasmids pBAD (arabinose...epitope tags useful for protein purification. pET LIC Series Various Plasmids T7-lacO (lactose/IPTG inducible...-PesaRlux controlling luciferase expression. pCW-LIC 26098 3x Tac promoter (hybrid of trp and lac promoters...Bacteria? E. coli are commonly used to store and replicate plasmids of all types, but beyond that, researchers...basic biology of a bacterial gene or redirecting metabolic pathways to produce a therapeutic compound, there...protein of interest. RK2 origin suitable for replication in many gram-negative hosts. pCri System Various...

...and applications.    Plant applications We outlined many synthetic biology MoClo applications, but...- and multi-gene constructs for a variety of applications. MoClo tools can be used in bacterial and cell-free...and the kits Addgene offers to kickstart these applications, no matter what your model organism is. MoClo... constructs that compose genetic circuits and metabolic pathways. For many MoClo systems, the individual...this blog, we will highlight some common MoClo applications and kits. Keep in mind that this technology ... it, MoClo might help you achieve it! MoClo applications and specialized tools Synthetic biology in bacteria...and multi-TU constructs for synthetic biology applications in bacteria or cell-free systems. The approach...

...genetic underpinnings of these processes has been complicated by the lack of precise genetic tools for modulating...recent advancements and offer commentary on their application to neuroscience research. What is CRISPR-based...accessible to neuroscientists, not all neuroscience applications require such optimized tools. This is elegantly...necessitates such molecular gymnastics for sensitive applications such as CRISPR screening. The plasmids used ...used by the Qi Lab for this particular application can be found here.  Why CRISPRa? Although cDNA overexpression...gene activation in select in vitro and in vivo applications, the tools described above make CRISPRa in the...relatively easy and accessible. There are also other applications for which CRISPRa is well-suited, including ...

... not need to be run in triplicates. ddPCR is an extremely accurate application, even in areas of the genome...blog post, we're going to focus on PCR-based applications (qPCR and ddPCR.) qPCR Quantitative/real-time...standard curves, and normal controls must be run in triplicate.  The nice thing about qPCR is that since it ...fluorescent probe and quencher to quantify specific amplicons.  Sybr has high background noise, due to the large... affected by AT-rich DNA and the length of the amplicon and it has a higher rate of false positives (Colborn...is not affected by AT content or length of the amplicon. As the probes can have several different markers...range of RQ values as noted above.   In both applications, results can be affected by DNA quality and ...

...few principles that will help you resolve most conflicts, even with difficult people. If I had to use one...strategies to become more assertive & resolve conflicts What Is Assertiveness? An assertive person is ... side-tracked, and you might create even more conflicts. 4) Define in advance how you would like the problem...in writing One of the most frequent sources of conflict is miscommunication. For example, you might misunderstand...you to resolve both personal and professional conflicts. At the workplace, these strategies will help ...miscommunication, which can lead to even more conflicts. If your supervisor is a difficult person, the...an inexperienced manager, you can resolve most conflicts if you communicate your ideas assertively, listen...

...- and ligation-independent cloning: A SLIC makeover In 2007, LIC received an important update, courtesy... and ligation-independent cloning (SLIC), eliminated many of LIC’s constraints by relying on the power...Ligation-independent cloning Ligation-independent cloning (LIC) was first developed in the 1990s. While traditional...transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs can hold a nicked...repairable by the bacteria after transformation. LIC is a reliable cloning method, but it is limited by...back the entire 10–12 bases. As such, the use of LIC is often limited to specially designed plasmids. ...of DNA!) Figure 1: Schematic of SLIC. To start the SLIC cloning process, a gene of interest is ...

...species or metabolic pathways. A new study takes us one step closer to the vision of metabolic biofactories...s vision of ‘biofactories’, most successes in metabolic engineering have been limited to the overexpression...be host-independent, beyond the oft published metabolic engineering realm of S. cerevisiae and E. coli...control via scRNA design is readily apparent in metabolic engineering. The authors elegantly demonstrated...demonstrated the potential of this to control a metabolic network in S. cerevisiae by using three RNA programs ...programs, the authors sequentially rerouted the metabolic flux to generate differently pigmented derivatives...regulatory switch. By no means limited to the field of metabolic engineering, this is such a powerful and flexible...