...Dutzler Labs EasyClone 2.0 Yeast Toolkit - Borodina Lab Golden GATEway cloning system - Wittbrodt Lab MultiSite...Vectors - Barbier Lab Cloning and Expression Bacteria and yeast expression vectors : FX cloning System - Geertsma...Parts Kit : Molecular cloning for E. coli - Densmore Lab EcoFlex MoClo : Modular cloning for applications ...- Lindquist Lab Synthetic Biology BIOFAB plasmid set : E. coli gene expression - Endy Lab CIDAR MoClo ... S. cerevisiae lab strains - Ralser Lab Return to top Resources for Microbiology General CRISPR...Freemont Lab MoClo-YTK : Assembly of single and multi-gene constructs for S. cerevisiae - Dueber Lab Yeast...Proteins FRET-based biosensors : E. coli expression - Pertz Lab Bacterial Fluorescence Labeling : Broad-Range...

...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...

...performing either PCR - or restriction enzyme -based cloning to test individual clones before use of more...restriction digests have become the most widely used method scientists employ to selectively move a specific...digested with restriction enzymes common to multiple cloning sites, will result in similar sized bands, thus...if you receive a plasmid from someone in another lab, or dig one out of the freezer and you are not 100%...Insert Orientation by Restriction Digest If you clone an insert into a vector by a single restriction ... enzyme, you will need to verify that it has be cloned in the correct orientation - this can be done by...restriction digest. Although this is never an ideal cloning strategy, sometimes it cannot be avoided. If you...

...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 ...

... sites to flank your insert : You can use PCR Based Cloning and add restriction sites to the ends of your... Protocols Subcloning Plasmid Cloning by Restriction Enzyme Digest (aka Subcloning) You may also like...Information about plasmid cloning by restriction enzyme digest (subcloning), including design and experimental...Reference . Background Subcloning by restriction digest is a commonly used lab technique. For the purposes...your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid...test digest later to verify that the insert was cloned in the correct orientation. If you cannot find ...of your recipient plasmid using Annealed-oligo Cloning . If you are lucky enough to have multiple options...

...pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty information Back to Top A. pLKO.1-TRC Cloning Vector A...these bases are important for cloning the oligos into the pLKO.1 TRC-cloning vector. Forward oligo: 5’ CCGG...GTATAGCTTAACGTAGGCATT 3’ Back to Top C. Cloning Oligos into pLKO.1 The pLKO.1-TRC cloning vector contains a 1.9kb stuffer...minutes at 95°C in a PCR machine or in a beaker of boiling water. If using a PCR machine, incubate the...digests. Digest pLKO.1 TRC-cloning vector with AgeI. Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep...Sequence of pLKO.1 TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning vector. The vector...working with the TRC to make this shRNA cloning vector available to the scientific community. Please cite...

...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 ...

... of interest are cloned into a Firefly luciferase reporter vector via Gateway cloning to test for the ... : Luminescence-based protein-protein screening method for the systematic and quantitative analysis of...Renilla and Firefly luciferase. Plasmids are Gateway cloning compatible. Learn more about DULIP in our 27 Hot... of false positives with the original STARR-seq method. Learn more about STARR-seq in our 27 Hot Plasmids...collection of empty backbone plasmids into which you can clone your regulatory element or gene of interest into... of 5' promoter/enhancer regions using Gateway cloning. Lentiviral. Jorge Ferrer 87067 pcDNA3.1-ccdB-Nanoluc...Creation of N-terminal Nanoluc fusions using Gateway cloning Mikko Taipale 87075 pLenti6.2-ccdB-Nanoluc NanoLuc...

...sequences are now available and more are currently undergoing quality control analysis in our lab and will be...Material. Rauch J, et al. bioRxiv 2020.04.20.052159 . Jennifer Doudna's lab has utilized Cas12a’s non-... the method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR). Mammoth Biosciences has ...reporter of SARS-CoV-2 protease Mpro now available from Xiaokun Shu's lab. A rapid, highly sensitive and open-access...and Bst polymerase constructs now available from Andrea Pauli's lab. Neutralizing nanobodies bind SARS-CoV...window) CRISPR tools and reporters now available from Stanley Qi's lab. COVID-19 Diagnostic Toolkit Enzymes...plasmid from the Abudayyeh-Gootenberg lab. A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for...

...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...

...based on factors such as selectable marker or cloning method. When using CRISPR, you will need to express...Plasmid Promoter Cloning Enzyme(s) Validated In Resistance Co-expressed Cas9 Depositing lab Cas9 species ...immunoprecipitation (enChIP) using CRISPR. Biochem Biophys Res Commun. 2013 Aug 11. pii: S0006-291X(13...species = S. pyogenes (PAM = NGG) 42875 pCRISPR BsaI E. coli, S. pneumoniae Kanamycin none, need Cas9 plasmid...

... to conventional methods for genomic purification, CRISPR-based purification methods are more straightforward...PA, and biotin tags, or an anti-Cas9 antibody. The CAPTURE system, developed by the Jian Xu lab , utilizes...Addgene's guide to using CRISPR plasmids in your lab, from knockouts to pooled library screening. Science...researchers to target anywhere from 2–7 genetic loci by cloning multiple gRNAs into a single plasmid — though some...Cas9’s proofreading capabilities. No matter the method, increased fidelity enzymes generate less off-target...happens. CASANOVA , developed by Dominik Niopek’s lab , is a way to optogenetically control genome editing...AcrIIA4 with the photosensitive LOV2 domain . Several labs have also controlled Acr activity with endogenous...

...using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA ...cell population. The region of interest is PCR amplified, PCR products are denatured-renatured, treated... The PCR product is then run on an agarose gel to determine whether the edit was successful. PCR amplification...screening individual clones. PCR amplification and next-generation sequencing (for HDR or NHEJ): Provides...targeted alleles. Involves PCR amplification of targeted region from DNA, subcloning into a plasmid, and screening...engineering using CRISPR-CAS9 system. Methods in Molecular Biology . 1239 , 197–217. PMID: 25408407 Hashimoto...selectable marker (drug or fluorophore) Delivery method Expression System Components of System Application...

...Plasmid 101: Gateway Cloning ) Gateway® cloning is a recombination based cloning method. The benefit of Gateway...Toposiomerase based cloning, often called TOPO® cloning or TA cloning, is a method that relies on the hybridization... molecular cloning techniques, including information on seven commonly used cloning methods. Molecular... most popular cloning methods used to create recombinant DNA. Restriction Enzyme Cloning Restriction enzyme...(endonuclease) based molecular cloning is the "classic" cloning method, and for many reasons, remains ... compared to other cloning methods. You can find a protocol for restriction cloning and an in-depth breakdown...101: Restriction cloning ) Back to Top Gateway® Recombination Cloning Gateway cloning (Image from Plasmid...

...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...

... the lab. Below are some common plasmid types: Cloning Plasmids - Used to facilitate the cloning of DNA... help you choose the right cloning method Plasmids 101: Restriction Cloning Plasmids 101: Inducible Promoters...the molecular biologist’s toolbox. To learn more about different types of cloning methods check out our...many different lab techniques that are important in molecular biology and plasmid cloning. Check out our...thus termed clones of the original cell. The plasmid DNA is similarly referred to as cloned DNA, and this... is known as DNA or molecular cloning . The process of molecular cloning enabled scientists to break chromosomes...optimized to help in the initial cloning of a DNA fragment. Commonly used cloning vectors include Gateway entry...

...plasmid is compatible with cloning methods available in your lab. When cloning your plasmids, be sure to...production. Created with BioRender.com. AAV Vector Production Cloning Cloning your transgene, gRNA, or...transfer plasmid can be done with most standard cloning methods, including restriction enzyme, Gibson Assembly...sites or may only be compatible with certain cloning methods (such as a Gateway destination vector), so ...more information on cloning and working with plasmids, visit Addgene’s Molecular Biology Reference . Production...mutations or insertions in the Cap region. Several methods have been developed and successfully employed to...and a systemic AAV capsid could avoid invasive methods such as intracranial injection. Systemic capsids...

... to conventional methods for genomic purification, CRISPR-based purification methods are more straightforward...PA, and biotin tags, or an anti-Cas9 antibody. The CAPTURE system, developed by the Jian Xu lab , utilizes...Addgene's guide to using CRISPR plasmids in your lab, from knockouts to pooled library screening. Science...researchers to target anywhere from 2–7 genetic loci by cloning multiple gRNAs into a single plasmid — though some...Cas9’s proofreading capabilities. No matter the method, increased fidelity enzymes generate less off-target...happens. CASANOVA , developed by Dominik Niopek’s lab , is a way to optogenetically control genome editing...AcrIIA4 with the photosensitive LOV2 domain . Several labs have also controlled Acr activity with endogenous...