... 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... 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....

...technology and it's applications.... Genome Engineering Videos CRISPR Technology and Applications CRISPR-Cas Technology...Technology and its Applications Related videos Genome Engineering: Basic Principles Genome Engineering... If you have trouble loading this video, please click here . Please note: the chimeric RNA work (10:25...

...Polyhedrin pFastBac LIC and pFastBac Dual LIC - Vectors from the Gradia lab pFB-LIC-Bse - Insect... expression, Ligation Independent Cloning (LIC) Bacteria Lac, T7, araBAD p15TV-L - His-tagged ... Arrowsmith Lab Plasmids - More LIC bacterial cloning vectors pTD plasmid... GFP for mammalian expression pcDNA3 GFP LIC cloning vector - C-terminal GFP ...

...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...

... 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...

...mCitrine 516 529 59 5.7 Monomer (A206K) pcDNA3 mCitrine LIC cloning vector (6C) - Mammalian Expression pCS2+8CmCitrine...Zebrafish/Xenopus/C.elegans/Sea urchin pET mCitrine LIC cloning vector (u-mCitrine) - Bacterial Expression...mOrange 548 562 49 6.5 2.5 hr Monomer pET mOrange LIC cloning vector (u-mOrange) - Bacterial Expression...mCherry 587 610 16 4.5 15 min Monomer pcDNA3 mCherry LIC cloning vector (6B) - Mammalian Expression pLV-mCherry...Zebrafish/Xenopus/C.elegans/Sea urchin pET mCherry LIC cloning vector (u-mCherry) - Bacterial Expression...

...Parkinson's Pablo Rivera-Fuentes 111723 pBacMam2-DiEx-LIC-N-flag_huntingtin_full-length_Q23 HTT Flag CMV, P10...Huntington's Cheryl Arrowsmith 111724 pBacMam2-DiEx-LIC-N-flag_huntingtin_full-length_Q73 HTT Flag CMV, P10...Huntington's Cheryl Arrowsmith 111725 pBacMam2-DiEx-LIC-N-flag_huntingtin_full-length_Q79 HTT Flag CMV, P10...Huntington's Cheryl Arrowsmith 111726 pBacMam2-DiEx-LIC-C-flag_huntingtin_full-length_Q23 HTT Flag CMV, P10...Huntington's Cheryl Arrowsmith 111727 pBacMam2-DiEx-LIC-C-flag_huntingtin_full-length_Q54 HTT Flag CMV, P10...Huntington's Cheryl Arrowsmith 111728 pBacMam2-DiEx-LIC-C-flag_huntingtin_full-length_Q73 HTT Flag CMV, P10...Huntington's Cheryl Arrowsmith 111729 pBacMam2-DiEx-LIC-C-flag_huntingtin_full-length_Q79 HTT Flag CMV, P10...

.... Feng Zhang LIC TAL Effector Assembly Kit Assembly by ligation-independent cloning (LIC). Validated in...Golden Gate TALEN 2.0 48705 pTAL7a Boris Greber Application of TALEN technology in hPSCs pTAL7a and pTAL7b...

...bacteria for expression and purification ( pET28a-LIC or other pET vectors are recommended). Open Enzymes...

...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...

... 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...

...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 ...

...complex loads the helicase onto the replication forks at either end of the origin. The helicase proceedes to...Plasmids utilize their host cell's replication machinery in order to replicate. As described in our previous...previous Origin of Replication post, DNA replication is initiated at the ORI and may be synchronized with the... for initiation of replication as the bacterial chromosome. Chromosomal replication in bacteria is carefully...the entire round of replication). Since the primary control point of DNA replication in bacteria is initiation...the DNA strands. Two replication forks are created by the action of the helicase and move in opposite ...once the replication forks reach each other on the opposite end of the chromosome, the helicases are released...

...ligation-independent cloning (SLIC), eliminates many of LIC’s constraints. Key to SLIC is the power of homologous...- and ligation-independent cloning: A SLIC makeover In 2007, LIC received an important update, courtesy...Ligation-independent cloning Ligation-independent cloning (LIC) was first developed in the 1990s. While traditional...restriction enzyme cloning used short sticky ends, LIC employed the exonuclease activity of T4 DNA polymerase...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. ...requirement for carefully designed DNA overhangs used in LIC.  As long as there was enough sequence homology (...