...through time, Christopher Marx's lab has engineered and validated a new method called FREQ-Seq. This strategy...ACS chemical biology 11.1 (2015): 31-37. pCri system The Gomis-Ruth lab designed the pCri system to simplify...can order from the repository: FX Cloning Kit BIOFAB Plasmid Set pCri System Cell Free Protein Expression...expression in bacteria, researchers at the BIOFAB and the Endy Lab created this kit. The plasmids within it...cyanobacterium Synechocystis sp. PCC 6803." Biology open (2016): bio-017129. Hsia, Justin, et al. "Modular...The Alexandrov lab at the University of Queensland’s Institute for Molecular Bioscience has provided a...please write to us in the comments section.  FX cloning kit This kit is designed to be used with Type IIS...

... of this method, isolation of a specific genomic region was an intensive process. One method required ...immunoprecipitation: a method for isolating specific genomic regions." Journal of bioscience and bioengineering 108.5 ...region In 2013, the Hodaka Fujii Laboratory first described a method to purify a specific genomic region...important discoveries in various biological fields. In 2015, the Fujii Lab identified non-coding RNAs associated...this site (Fujita and Fujii, 2013). They call this method engineered DNA-binding molecule-mediated chromatin...Zinc-finger or TAL proteins, can be used. The Fujii lab demonstrated this in mammalian cells by expressing...bound genomic locus, which they then confirmed by PCR. Subsequent mass spectroscopy studies allowed for...

...G418 selection, individual gene edited clones are identified by PCR. Then, if necessary for subsequent experiments...the homologous recombination pathway. The Waldman lab at the Georgetown University School of Medicine has...the past fifteen years. Some examples from their lab include using AAV to introduce epitope tags into ...suppressor in human cells (Kim et al, 2016). The Waldman lab has designed their AAV tagging vectors (pAAV-SEPT-Acceptor...these tagging vectors for your specific gene, the lab recommends designing homology arms with the modification...synthesis company such as IDT or Genscript, and cloning them sequentially into the chosen acceptor vector...knockin efficiencies of 1-40% with G418-selected clones. However, there are important disadvantages to ...

...enriched via biotin pull-down methods and identified using mass spectrometry. The Roux Lab’s new and improved...process of cloning, combining, and testing these different parts can be quite arduous. The cloning process...Integration of Design Automation Research lab Moduclar Cloning (CIDAR MoClo) Parts Kit. This Kit consists...interest and cloned into provided destination vectors using low-cost, one-pot golden gate cloning that results...Iverson, et al. ACS Synth Bio. 2015. PubMed PMID: 26479688. RAS Pathway Clone Collection 2.0 Ras proteins...compatible with both standard Gateway cloning and the FNLCR Combinatorial Cloning Platform (CCP); the latter of...minimize off-target effects. The library was cloned into the Liu lab’s own pAc-sgRNA-Cas9 insect expression backbone...

...multicistronic element and reporter cloned in. In these plasmids you would simply clone your gene of interest into...replaced.  Additionally, 2A peptides could be PCR-cloned between your genes of interest and you can then...Addgene's Plasmid Guide for more molecular biology and cloning information. ...interest, scientists have instead developed novel methods to co-express your gene along with a reporter, ...the experimental cell type and the specific genes cloned into the vector.3  2A Peptides To overcome some...into the multiple cloning site up or down stream of the IRES or 2A element (depending on the placement ...you can start with a plasmid that has multiple cloning sites flanking the multicistronic element(s), or...

...or dip transformation method. The general steps for this method include: Cloning and transforming a plasmid...following floral dip transformation’, Plant Methods. BioMed Central, 2(1), p. 19. doi: 10.1186/1746-4811...my favorites. Plasmids from the Tsuyoshi Nakagawa lab are binary gateway vectors and can be used for promoter... there are a series of plasmids from the Dinneny lab that additionally allow for visual selection of transformants... Weigel and Glazebrook, 2002. Tips: Do a colony PCR on your agrobacterium: Getting a transformant takes...remaining starter culture can be used for colony PCR and glycerol stocks. Dip/drip plants more than once...Harrison, S. J. et al. (2006) ‘A rapid and robust method of identifying transformed Arabidopsis thaliana...

...degraded. Cloning Grade DNA (high quality/low quantity) – You can usually complete a cloning experiment...s no secret that labs produce a lot of waste. If you are looking to reduce your lab’s environmental impact...option is to use the kit that is already available in your lab or the one you are most familiar with. And...options Manufacturer DNA Isolation Method Kit Sizes Available Standard Miniprep Cost Reagents/Columns...Automation Eco-friendly Kits Specialty Kits Available Bio-Rad Column Mini, Midi $$ No No Not mentioned...DNA quality can inhibit important reactions like PCR or restriction digests. Transfection Grade DNA (high... salts, ethanol, or endotoxins. The most common method for checking both quantity and quality of your ...

...DNA cloning and assembly, New England Biolabs, Inc.         Eric Cantor is a molecular biologist and...Single insert cloning efficiencies were measured based on the acquisition of antibiotic selectable markers...the destination plasmid. Antibiotic selection allowed for high throughput cloning efficiency measurements...suppression of background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion...While T4 DNA Ligase, the mainstay of most biotechnological cloning efforts for over 50 years, prefers ligation...other plasmid cloning techniques Resources on Addgene.org Find basic molecular biology protocols Find...Find plasmid cloning protocols Check out our molecular biology reference pages ...

... follows a simple workflow: Clone spacer(s) into the CRISPR array Clone desired DNA cargo into the mini-transposon...the Sternberg Lab at Columbia University Medical Center. He currently studies CRISPR biology and CRISPR ...blogger, Leo Vo, a PhD candidate in the Sternberg Lab at Columbia University Medical Center. DNA transposons... for genetic studies (e.g. using mariner), as a method of tagmentation for next-gen sequencing pipelines...of CRISPR-transposons by the Sternberg and Zhang labs (Peters et al., 2017; Klompe et al., 2019; Strecker...CRISPR-transposon systems developed by the Sternberg lab derives from Vibrio cholerae (V. cholerae INTEGRATE... in E. coli (Klompe et al., 2019). Recently, the lab introduced a streamlined expression approach (Fig...

... Inc. Plasmids and PCR products are the bread and butter of molecular biology labs the world over. Scientists...historically matched the needs of most molecular biology labs. Recent and rapid advances in technologies that...(NGS) sequencing have prevented laboratories from using these methods for routine processes like plasmid... the feasible size and scope of projects in many labs. However, the high costs of sample preparation for... problematic for conventional NGS library prep methods because the yield or quality of individual libraries...of non-plasmid DNA or heterogeneous and/or mixed clones present in the original sample. At seqWell, we ...subjected to a DNA-barcoding step that uniquely labels DNA from each well of a 96-well plate. Then, samples...

...The Marraffini lab and Qi lab developed early systems for E. coli; while the Marraffini lab used a native...recombineering-based MAGE The Nielsen lab’s CRMAGE system is a fast, multiplexable method that combines CRISPR and ...the Sternberg and Zhang labs developed CRISPR-transposons. The Sternberg lab's system called INTEGRATE...other parameters. .  Stanley Qi lab's CRISPRi plasmids Stanley Qi’s lab demonstrated that a minimal, regulatable.... Marraffini lab's CRISPRi/a plasmids In addition to the early work of the Marraffini lab pioneering genome...pneumoniae. Robert Husson lab's M. tuberculosis CRISPRi plasmids A group from the Husson lab used CRISPRi to study... search for a CRISPR activation method in E. coli, Jesse Zalatan’s lab used an approach to identify known...

...protein? Classical methods for studying protein phosphorylation A number of classic methods exist for this... be cloned with an ORF of interest for customized experiments. Once the ORF of interest is cloned into...for use by the Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains...reference ‘Rinehart Lab reagents for improved expression of recombinant phosphoproteins’ available on the Addgene...Colony PCR Read about Tags for Protein Purification Resources on Addgene.org Find Synthetic Biology Plasmids...such as phosphospecific antibodies, which aren’t available for the majority of documented phosphorylation...translation. Many ‘flavors’ of Sep-OTS plasmids are available through Addgene, but we have worked most extensively...

...uses base editors and HDR methods for rice crop improvement in the laboratory of Yinong Yang at PSU.   ...of base editors for plant systems. Thus, the Yang Lab at The Pennsylvania State University (PSU) is adapting... al., 2017). Our vectors have two Bsa1 sites to clone the single guide RNA (protospacer+scaffold) in a...single polycistronic tRNA-gRNA (PTG) gene system to clone multiple protospacers which enables us to target...restriction enzyme (RE) recognition site, RE analysis of PCR product could be used to detect success. Although...mutation by an adenine base editor would restore antibiotic resistance. To find such a protein, they created...base editors for plant There are several CBEs available for plant systems. Target-AID based (CBE-2)-  ...

... a robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol. 19 (2018... has remained a challenge. Enter Easi-CRISPR: a method that harnesses the power of ssDNA donor molecules...60%. This is much more favorable than previous methods yielding 1-10% knock-in. Read on to learn how you...mouse embryonic stem cells. After selecting for clones with the recombined product, you’d microinject ...DNA, but Miura et al. (2015) developed the ivTRT method to ameliorate this problem. Briefly, you design...with standard microinjection and electroporation methods using in mouse transgenics. Designing an Easi-CRISPR... the insertion site. If you are using the ivTRT method, try to make the first two bases of the left homology...

...drawbacks of different methods (for comprehensive protocols for all of the methods discussed here refer ...depending on the vector backbone. When using qPCR based methods for viral titration consider using a reverse...treated with the antibiotic, and colony-forming units quantified. While these methods tend to be more ... expression. The most accurate method of lentiviral titration uses qPCR to measure the number of proviral...Comparison of lentiviral vector titration methods."BMC biotechnology 6.1 (2006): 1. PubMed PMID: 16836756....in a matter of hours. A major drawback of this method, however, is that a p24 ELISA measures all p24 in... direct p24 measurement. Like a p24 ELISA, this method is relatively quick, providing results in less ...

...widely used qPCR and antigen tests.  Labs including Jennifer Doudna’s lab and Feng Zheng’s lab had already...interfere with it in a BSL-2 lab instead of a BSL-3 lab. Jesse Bloom’s lab published a protocol and reagents...Hyeryun Choe’s lab identified that ACE2 is the functional receptor for SARS-CoV. The lab also found that...openly available including those from Andrea Pauli and Julius Brennecke. They developed a method that uses...19 related plasmids. These plasmids have reached labs in over 75 countries. Figure 1: SARS-CoV-...been used to accelerate COVID-19 research in other labs. Spike protein, variants, and pseudotyping The SARS-CoV...called HexoPro, was developed by Jason McLellan’s lab. This version of the spike protein was developed ...

...concentration. Three methods are broadly used for virus titration: the p24 ELISA dosing method detects the expression...transcriptase activity assay uses qPCR to quantify viral RNA. All of these methods have been described in a previous...non-dividing cells. To target these un-transfectable cells, labs use viral vectors and particularly retroviral vectors...plasmids The transfer plasmid is the vector you clone your gene of interest into and is an important feature...production and transduction Once you have cloned your gene of interest into lentiviral transfer vector, the...expression of viral capsid antigen p24, the FACS dosing method correlates viral titre to the number of cells transduced...with the virus in order to aid viral binding. This method, called spinoculation, has been found very effective...

...located within a cell. The Goedhart lab, Gadella lab, and collaborators have an ongoing effort to improve..., et al., bioRxiv. 2020. https://doi.org/10.1101/160374 Nanobodies- there's a simple method for that!...hPSCs), Lindy Barrett’s lab at the Broad Institute developed fluorescently labeled piggyBac vectors for ...Sanchez and Alice Ting. Sanchez MI et al., Nat methods. 2019. https://doi.org/10.1038/s41592-019-0665-... size, more durable, and usually much easier to clone, express, and purify compared to antibodies. Nanobodies...Hara, and Kazuo Umemura have described a simple method which alleviates the inclusion body problem and...transposase. Image from Hazelbaker et al., 2020. The lab tested this system by repressing and activating expression...

...the protocols provided by the depositing lab whenever available. Addgene also shares the protocols we’ve...can’t (or don’t want to) make viral preps in your lab, there are custom viral prep services that can prepare...the original library constructed by the depositing lab. (For our more popular libraries, you might receive...information or scripts provided by the depositing lab for data analysis.  I'm often asked whether this ... associated with the library and reading their methods sections to see what worked.  Ready, optimize, ... - An excellent protocol paper by the Feng Zhang lab describing construction, lentiviral production, transduction...CRISPR/Cas Systems Forum- Moderated by the Feng Zhang lab, this is a useful place to ask experimental questions...