...deletion clones with a proofreading polymerase and clone the amplicons ( e.g., with a PCR cloning kit) into...to perform PCR in step 6.3 . Assemble a 20 μl PCR with the following components: 10 μl 2x PCR mix, 0.5 ...CRISPR/Cas9 Clones for Deletions and Clone Selection For suspension cells, transfer all clones to a single... for cloning and expression purposes. NOTE: Here we discuss the usage of the pSpCas9(BB) plasmid (pX330...pX330) (Addgene plasmid ID 42230). This plasmid allows for the simultaneous expression of sgRNA and SpCas9...utilized, such as pX458 (Addgene plasmid ID 48138) or pX459 (Addgene plasmid ID 48139), which include GFP ...each well) to a 96-well PCR plate using a multichannel pipette. Centrifuge PCR plate at 400 x g for 5 ...

...alleles. Involves PCR amplification of targeted region from DNA, subcloning into a plasmid, and screening...Additionally, many plasmids containing validated gRNAs are now available through Addgene. These plasmids contain ...how to design your gRNA . Browse Plasmids: gRNAs Synthesize and clone desired gRNAs Once you have selected...inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, ...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 quantitative...

...co-transfect several plasmid together, with each plasmid having a targeting spacer cloned into the backbone...positive and negative clones. Clones with insertion will show only linearized plasmid of ~8.5 kb (only AgeI... may also like... Zhang Lab CRISPR plasmids General CRISPR Plasmids Dr. Le Cong, the first author of one...to add the CACC and AAAC cloning overhang. When designing oligos for cloning my target sequence into a...Cas9, clone two spacers for each locus into PX330, then co-transfect or delivery the two plasmids together... big concern, you could go ahead with PX330 plasmid, clone in your target guides, test run them and then...better for PCR and surveyor. I used DNA polymerase Takara Ex Taq ™ DNA Polymerase for my genomic PCR, but couldn't...

... Zhang Lab Plasmids The Human TFome Library Stem Cells Protocols & References MORF Plasmids MORF Pooled...TF ORF barcode PCR for NGS QC and bulk screening (PDF, 85 KB) TF ORF barcode dial out PCR for 10X scRNA-seq...scRNA-seq (PDF, 59.2 KB) Plasmids ID Plasmid Gene/Insert Pooled Library The MORF pooled library is a comprehensive...suggestions for other plasmids that should be added to this list? Fill out our Suggest a Plasmid form or e-mail...isoforms and two control vectors. The individual clones, or the pooled library containing the entire pooled...

...For cloning information, please view: Zhang Lab General Cloning Protocol 237.2 KB SpCas9 Plasmids The ...adenocarcinoma pathology. These plasmids as well as a backbone plasmid for cloning new targets are described...recombinase and an sgRNA backbone for cloning new targeted plasmids. The plasmid can be digested using SapI, which...2A-EGFP-KASH and an sgRNA backbone for cloning new targeted plasmids. The plasmid can be digested using SapI, which...tracrRNA: This plasmid contains three expression cassettes. To target a given site, the plasmid can be digested... for sgRNA cloning; has GFP-KASH for FACS sorting SaCas9 + single guide RNA: These plasmids contain two... used in the SpCas9 plasmid series. ), and a pair of annealed oligos can be cloned scarlessly into the...

...Protein Species Tag Donor Plasmid gRNA plasmid gRNA plasmid FLAG pFETCh_Donor CEBPB Human FLAG pFETCh_CEBPB...the remaining 5 plasmids include Cre and FLP expression vectors, a general cloning plasmid, and a prebuilt.... Multiple gRNA plasmids may be used. The HDR (homology directed repair) donor plasmid with homology arms...sequence, as well as design and clone in the homology arms for the donor plasmid. The Mendenhall and Myers ...arm cloning: Mendenhall & Myers FETCh-seq Protocol 134.7 KB Mendenhall and Myers Tagging Plasmids Protein...backbone to clone the left and right homology arms. More details on how to use this plasmid in your experiments...individual tagging event. The kit contains 26 plasmids; 21 of the plasmids are for use in SapTrap reactions for...

... on the plasmid. gRNA plasmids that do not co-express a Cas protein require a separate plasmid that does...Lab . Plasmid Expression System Cloning Enzyme Co-expressed Cas9 Cas9 System Selection PI pCRISPR 42875...gRNA Vectors CRISPR Plasmids: Empty gRNA Expression Vectors Browse CRISPR Plasmids By Function Genome ... these independent gRNA plasmids can be paired with a wide variety of plasmids and therefore are not limited.... Whether the plasmid contains Cas9 and if so, which function of Cas9. We have plasmids with Cas9 that... for Cloning 67720 Worm NotI/BamHI none S. pyogenes Colaiacovo pRB17 52527 Fly blunt end cloning none ...none, need plasmid 65775 N. meningitidis Joung VVT1 65779 Mammalian BsmBI none, need plasmid 65776 S. aureus...

...wide variety of Cas-containing plasmids. ID gRNA Plasmid Promoter Cloning Enzyme(s) Delivery Resistance...Cas9 plasmid Joung 46170 PU6::klp-12_sgRNA cU6 PCR (see paper) Transfection none, need Cas9 plasmid Calarco...gRNA expression plasmid to target the dCas9-activator to your specific locus. ID Plasmid Gene/Insert Promoter...Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using ... need Cas9 plasmid Boxem 47941 pMB60 T7 BsaI In vitro transcription none, need Cas9 plasmid Boxem 47912...elegans none, need Cas9 plasmid Ristow Do you have suggestions for other plasmids that should be added to...CRISPR plasmids that have been designed for use in C. elegans. CRISPR...

...List of plasmid kits and accessory plasmids available from Addgene for generating your own specific TALE...336 pFusX clones allowing for Golden Gate assembly of 15-16 RVD TALEN inserts in one cloning step rather...cells. Keith Joung Zhang Lab TALE Toolbox PCR/Golden Gate cloning method. Optimized for human expression....Yamamoto Additional Plasmids for Use with Addgene TALEN Kits For Use With: ID Plasmid Depositing PI Description... 2, 3, or 4. For both plasmids sequence positions 1214–2210 of pTAL3 were cloned into a pCS2 expression...putative enhancers in mammalian cells. These plasmids allow for cloning DNA fragments encoding TAL effector repeat...TALE Toolbox 49622 - 49647 24 plasmids Thoru Pederson TALEColor plasmids: destination vectors for use ...

...Description Plasmids in protocol Download protocol Church gRNA design and cloning gRNA cloning vector PDF... and cloning for Cas9 orthologs Cas9 plasmids PDF 104.1 KB Chen and Wente Zebrafish: gRNA cloning, in ...optimized Cas9 PDF 66.8 KB Fujii gRNA design and cloning gRNA cloning vector Retroviral vectors: neomycin (pSIR-neo...Fly: gRNA cloning pAc-sgRNA-Cas9 PDF 165.1 KB Marraffini Bacteria: pCas9 new spacer cloning pCas9 PDF ...Lander gRNA cloning into pLX-sgRNA pLX-sgRNA PDF 125.9 KB Sontheimer gRNA design and cloning All-in-one...All-in-one plasmid containing expression cassette for NmCas9 and BsmBI site for cloning in gRNA: pSimpleII-U6-...donor plasmid backbones pSL1180-HR-PUbECFP & pSL1180polyUBdsRED PDF 583.3 KB Zhang gRNA cloning CRISPR...

...Foundation Plasmids Drew Endy Lab Plasmids Jennifer Molloy Lab Plasmids Golden Gate cloning...collection include polymerase chain reaction (PCR), cloning, isothermal amplification, reverse transcription...Golden Gate assembly. Plasmids Explore the collection and order individual plasmids via the links below:...Browse the Open Enzyme collection of plasmids at Addgene for generating enzymes for molecular biology...the basis of manufacturing their own enzymes by cloning them into an expression vector of their choice ... (Link opens in a new window) . How to Use The plasmids in the Open Enzyme collection contain genetic ...not functional by themselves. Researchers should clone these enzymes into an expression vector of their...

... wide variety of Cas-containing plasmids. ID Plasmid Promoter Cloning Enzyme Co-expressed Cas9 Cas9 System...gRNA expression plasmid to target the dCas9-activator to your specific locus. ID Plasmid Gene/Insert Promoter...gRNA expression plasmid to target the dCas9-repressor to your specific locus. ID Plasmid Gene/Insert Promoter...Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using ...suggestions for other plasmids that should be added to this list? Fill out our Suggest a Plasmid form or e-mail...expression plasmids that have been designed for use in plants. CRISPR... CRISPR Plants CRISPR Plasmids: Plants Browse...

...luciferase. Plasmids are Gateway cloning compatible. Learn more about DULIP in our 27 Hot Plasmids from 2016...Collections Luciferase Plasmids Luciferase Plasmid Collection You may also like... Fluorescent Proteins Blog...luciferase reporter. These plasmids improve over the original STARR-seq plasmids by using the bacterial ORI... of interest are cloned into a Firefly luciferase reporter vector via Gateway cloning to test for the ...is a collection of empty backbone plasmids into which you can clone your regulatory element or gene of...otherwise noted, these plasmids are meant for expression in mammalian systems. ID Plasmid Luciferase Type(s...luciferase expression plasmids; these are noted with the viral service icon . ID Plasmid Luciferase Type Promoter...

... Plasmid Collections Microbiology Plasmids Microbiology Plasmids and Resources Addgene...Microbiology Resources Plasmid Kits for Microbiology : Sets of plasmids for fluorescence, cloning and expression...guide on assembly standards. Browse Plasmids by Microbe System Plasmids for Archaea Species Haloferax sp....Vibrio sp. Yersinia pestis Plasmids for Cyanobacteria Cyanobacteria Plasmids for Fungi Species Aspergillus...Aspergillus sp. Cryptococcus sp. Plasmids for Prions Prions Plasmids for Protozoa Species Entamoeba histolytica... tools , or by suggesting plasmids for the collection. Return to top Plasmid Kits for Microbiology Kits...Kits are sets of plasmids intended for use together (e.g. cloning and expression systems) and therefore...

...Reporter Plasmids You may also like… Plasmids 101 Addgene's Molecular Biology Guides Plasmids 101: E. ...interest. Plasmid ID Promoter Inducer(s) PI Purpose Murray Lab pBEST plasmids Various Plasmids pBAD, OR2...reporter gene. Plasmid ID Promoter Reporter PI Purpose pRU Series Various Plasmids Cloned by researcher...Purification Controlled Expression Reporter Plasmids Reporter Plasmids Tagging and Visualization Purification...list of our bacterial CRISPR plasmids, see here . pGRG Series Various Plasmids Tn7 Transposition Nancy Craig...Craig A gene of interest cloned into one of these plasmids can be transposed into the attTn7 attachment...subtilis and P. pastoris . All plasmids use the same restriction enzymes for cloning so multiple constructs can...

...wide variety of Cas-containing plasmids. ID gRNA Plasmid Promoter Cloning Enzyme(s) Validated In Resistance...efficiency than NHEJ. ID Plasmid Gene/Insert Promoter PI Publication Browse CRISPR Plasmids By Function Genome...gRNA expression plasmid to target the dCas9-activator to your specific locus. ID Plasmid Gene/Insert Promoter...gRNA expression plasmid to target the dCas9-repressor to your specific locus. ID Plasmid Gene/Insert Promoter...Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using ...Ampicillin none, need Cas9 plasmid Qi Do you have suggestions for other plasmids that should be added to ...PAM = NGG) 42875 pCRISPR BsaI E. coli, S. pneumoniae Kanamycin none, need Cas9 plasmid Marraffini 42876...

...nucleocapsid protein (Brian Geiss). Plasmids SARS-CoV-2 Plasmids Many plasmids containing SARS-CoV-2 sequences...Addgene will maintain this plasmid collection page, which highlights plasmids available that may be useful...SARS-CoV-2 nucleocapsid protein. SARS-CoV-2 Plasmids : Plasmids that are available or coming soon containing...containing SARS-CoV-2 sequences. SARS-CoV Plasmids : Plasmids that are available containing SARS-CoV (SARS ...to popular envelope and packaging plasmids. Ginkgo Bioworks Plasmid Collection - Addgene has partnered...Bioworks to distribute SARS-CoV-2 plasmids. The collection contains: Plasmids for E. coli and Yeast expression...available Ginkgo SARS-CoV-2 plasmids are curated on a separate Ginkgo Bioworks Plasmid Collection page. SARS-CoV...

...DNA encoding zinc finger arrays should be cloned into plasmids also available through Addgene--the specific... Plasmid Collections Zinc Finger Consortium Zinc Finger ...cCGCTTCAGCggtctGTGGACTCGa zebrafish similar to grp151 (or gpcr-2037) OZ535 and OZ536 OPEN OPEN aGCCATCATCaggttcaGGATGAGCCc...

... Plasmid Collections Cre-lox Plasmids Cre-lox Plasmids Additional...Cre-lox Plasmids Addgene's Blog: Cre-lox Addgene's Blog: FLEx Vectors Cre-containing Plasmids loxP Constructs...viral preparations of plasmids that are currently in our repository. ID Plasmid Description Promoter Expression...loxP plasmids, reporter genes indicate which cells have undergone Cre recombination. These plasmids can...efficiency, selecting desired clones, and making transgenic mice. ID Plasmid/Article Description Expression...suggestions for other plasmids that should be added to this list? Fill out our Suggest a Plasmid form or e-mail... Cre-lox plasmids for your experiments. Plasmid...

...anywhere from 2–7 genetic loci by cloning multiple gRNAs into a single plasmid — though some have achieved ...isolate clones containing the desired edit (see: Validate genetic modification ). Browse Plasmids: Endogenous...the genomic DNA. This sequence is cloned into a gRNA expression plasmid but does NOT include the PAM sequence...Addgene's guide to using CRISPR plasmids in your lab, from knockouts to pooled library screening. Science... cell must be validated experimentally. Browse Plasmids: Double-Strand Break (Cut) Multiplex Genome Engineering...cases, delivering multiple gRNAs using a single plasmid ensures that all gRNAs will be expressed in the...likelihood that any cell containing the CRISPR plasmid will have all desired genomic edits carried out...