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  1. Evolution of Lab Techniques

    Type
    Blog Post
    ...replaced by real-time PCR to answer the same experimental questions. Real-time PCR detects and quantifies...rate than real-time PCR (4). Restriction mapping and modern sequencing Restriction mapping informs on .... Southern blotting and quantitative real-time PCR Southern blotting, named for its inventor Edward ...than just at the end as in standard PCR. This allows the researcher to compare different DNA sequences...requires a large amount of high-quality DNA, real-time PCR has several advantages including easier automation...Speleman, and Jo Vandesompele. "Real-time quantitative PCR as an alternative to Southern blot or fluorescence...Sequencing Results Check out Plasmid Cloning by PCR Resources on Addgene.org Check out Our Protocol Pages Find...
  2. Ligation Independent Cloning

    Type
    Protocol
    ...sterile dH20 to 40 μl Step 4: Amplify Insert by PCR Perform PCR amplification of your insert following the...ensure a melting temperature between 50-60°C for your PCR primers. Step 2: Linearize Vector In this example...like this: Note: Elute your purified digestion and PCR products with sterile water (instead of TE buffer...important to remove all free nucleotides from your PCR product before proceeding, as they will interfere...Step 5: Create Insert Overhangs Treat your purified PCR product with T4 Pol in the presence of dCTP (not ...Empty vectors for LIC typically employ type II restriction enzymes (e.g. BsaI), which cut at a specified...overhangs with a single enzyme, and remove the restriction sites from the final product (no "cloning scars...
  3. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    ...primers are amplified and used to generate Illumina sequencing libraries using a 2-step PCR-based protocol...with Type IIS restriction enzymes and standardized primers to clone your gene of interest into both sequencing...biology 11.1 (2015): 31-37. pCri system The Gomis-Ruth lab designed the pCri system to simplify the protein...protein of interest. The plasmids used to generate these fusions all use the same restriction enzymes for...the repository: FX Cloning Kit BIOFAB Plasmid Set pCri System Cell Free Protein Expression Kit FREQ Seq... pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance... they are unlikely to be found in your gene of interest) and prevent incorporation of extensive sequences...
  4. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...plasmid using restriction enzymes Watch the Video! Polymerase Chain Reaction (PCR) Basic PCR protocol with... Video Restriction Cloning Assemble plasmids by restriction digest and ligation Cloning by PCR Generate...CRISPR Library Amplification Amplify CRISPR pooled-plasmid libraries Diagnostic Restriction Digest Verify...Generate restriction sites by PCR Modification by Annealed Oligo Cloning Add a short stretch of DNA to a plasmid... spectrophotometer Restriction Digests Cut and analyze plasmid DNA with restriction enzymes Agarose Gel...Watch the Video! AAV Titration by qPCR Using SYBR Green Technology Use qPCR to measure the physical titer ...preparation Watch the Video! AAV Titration by ddPCR Use ddPCR to measure the physical titer of your adeno-associated...
  5. Tag Your Favorite Yeast Genes with Ease

    Type
    Blog Post
    ...protocol is surprisingly simple, requiring only a PCR product containing the modifying sequence flanked...to the chromosomal site of insertion. The linear PCR product is introduced into the cell by direct transformation...selection marker (KanMX, HphMX, or His3MX).  Each PCR product from this collection will yield an insert...multi-color imaging experiments. Interested in epitope tags? Others may be interested in attaching epitope tags...tags have been integrated. Simply design your amplification primers with the desired targeting homology—... fluorescent proteins (FPs) to a yeast gene of interest. These fluorescently tagged genes are expressed...tags to their genes of interest, allowing for easy capture and detection of proteins and complexes, without...
  6. Plasmids 101: Golden Gate Cloning

    Type
    Blog Post
    ...original plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden Gate ...this is to "domesticate" your fragment: PCR-based amplification can be used to create silent point mutations...s) thus eliminating these from your gene of interest. PCR products are then digested with the Type IIS...relies on Type IIS restriction enzymes, first discovered in 1996. Type IIS restriction enzymes are unique...If your genes of interest or destination vector contain multiple internal restriction sites that may not...: Sequential amplification of cloned DNA as tandem multimers using class-IIS restriction enzymes. Lee ...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method has...
  7. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...ORFs from an ORFeome Gateway library or any PCR-amplified ORF cloned into pDONR221 can be expressed either...antibiotic cassette to the gene of interest. The gene to be expressed is amplified using a gene-specific forward...ribosome binding site, SacI restriction site and a linker region. The PCR product is then ligated into...by scFv antibodies fused to sfGFP. This system amplifies the intensity of fluorescence signal and enables...can occur in this system if a small contaminating PCR product formed from primer dimers and containing ...cognate peptide epitopes fused to the protein of interest are recognized and thus fluorescently labeled ...modified to use different antibiotic resistances, restriction enzymes and plasmid backbones. pUCXKT contains...
  8. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    ... showed correctly sized inserts following colony PCR of 10-20 colonies using primers bordering the multiple...that is refractory to enzymatic manipulation, amplifying phage DNA with phi29 polymerase prior to fragmentation... whole genome amplification (by a mechanism known as multiple displacement amplification) even in the ...recognize on a host cell. To make matters even more interesting, much of what a phage faces as it approaches ...abilities (6). This can be accomplished using a restriction enzyme with a short recognition sequence (like...with incubating this membrane with your cells of interest and growing them up overnight. Assuming they expressed...that their host colonies grew. If your cells of interest stick to the same location as a given protein,...
  9. AAVs CREATed for Gene Delivery to the CNS and PNS

    Type
    Blog Post
    ...inversion creates a template that’s suitable for PCR amplification using pre-designed primers. By using this ...infected the cell type of interest. Repeat rounds of infection and screening by PCR enrich for capsid variants...those that cross the barrier can be identified by PCR later. The PNS presents a different problem: it’s...infect an animal expressing Cre in a cell type of interest, a scientist can isolate DNA from the infected...with increased ability to infect the cell type of interest, which, in the case of Chan et al, was neurons...of a transgenic mouse. 2. Cell-type-specific restricted transgene expression in the CNS and PNS Previously...promoter or enhancer now allows for cell-type-restricted expression in both the CNS (figure 5 in Chan ...
  10. Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

    Type
    Blog Post
    ...oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) method to suppress PCR amplification of specific DNA sequences...molecules associated with specific genomic regions of interest in a non-biased manner. To achieve this goal, ...
  11. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    ...using BsaI, and promoter-gRNA fragments are PCR amplified for cloning into one of three Zeamays codon-...the first gRNA using restriction enzyme BbsI and the second gRNA using restriction enzyme BsaI. If you’... of gRNAs Golden Gate Assembly uses Type IIS restriction enzymes, which cleave outside of their recognition...These expression vectors each contain Type IIS restriction sites flanking the promoter-gRNA construct, but...oligonucleotides. These plasmids contain Type IIS restriction sites that flank the promoter-gRNA constructs...
  12. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...Reaction (PCR) by using a biotinylated primer. Biotin can also be enzymatically incorporated during PCR using...complexes bound to your protein of interest leading to amplification of the reporter signal and easier ...macromolecule of interest To utilize the natural binding of biotin and avidin, macromolecules of interest first ...secondary antibody that targets your protein of interest or primary antibody respectively. Avidin bound...sites found on avidin are also ideal for signal amplification during these experiments. During detection, ...methods to chemically biotinylate macromolecules of interest both in vitro and in vivo. Biotinylation reagents...AviTag. Using AviTag, one can label a protein of interest with biotin by simply creating a fusion in which...
  13. CRISPR Plasmids - Tagging

    Type
    Collection
    ... in the following articles provide PCR templates for amplification of the tag (eg GFP, Flag, YFP, etc)... technique for use in Drosophila cells that uses PCR to generate both an expression cassette for the Cas9...plasmids for endogenous tagging of your gene of interest. CRISPR... design. How to use CRISPR to tag your gene of interest Mendenhall and Myers Tagging System The Eric Mendenhall...plasmids as listed in each row. If your own gene of interest is currently unavailable, you will need to design...expresses Cas9, PITCh-gRNA, & FBL-specific gRNA pCRIS-PITChv2-FBL - PITCh donor vector for EGFP-2A-PuroR...
  14. 6 Tips for Analyzing and Troubleshooting Sanger Sequencing Results

    Type
    Blog Post
    ... requires you to perform your own Big Dye PCR amplification reaction (as opposed to using the all inclusive... from your sequencing runs.   When you run a restriction digest on a gel you always include proper controls...primer at least 50bp upstream of the sequence of interest. 5. Use a silica spin column for purification ...
  15. What's the Best Way to Elute and Store Your Plasmid DNA?

    Type
    Blog Post
    ...degrade the double helix, fragmenting plasmid DNA, PCR product, or chromosome. One of the main causes of...most common E. coli strains used for plasmid amplification are endA negative, meaning the gene has been... tools used in modern molecular biology like restriction enzymes and polymerases. In the situation where...
  16. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...SLIC cloning. Amplify the pCoofy vector and the sequence of gene of interest in separate PCR amplication...during the PCR amplification step, so that only vectors with the desired sequence of interest are retained...method for protein inactivation Your protein of interest does x, but it also participates in y. Depletion...accomplished by knocking down your endogenous protein of interest and co-expressing its replacement recombinant ...into Golden Gate entry vectors via traditional restriction enzyme cloning, TA cloning, or annealing of oligonucleotides...would like to extend this work into their RTK of interest. Grusch, M. et al., EMBO J 2014 Aug 1;33(15)... genetic tag which can be fused to proteins of interest for EM imaging, or targeted to subcellular regions...
  17. Plasmids 101: What is a plasmid?

    Type
    Blog Post
    ... sequence used as an initiation point for PCR amplification or sequencing. Primers can be exploited for... Short segment of DNA which contains several restriction sites allowing for the easy insertion of DNA....“vectors” or “constructs.” To insert a gene of interest into a vector, scientists may utilize one of a... a variety of cloning methods (restriction enzyme, ligation independent, Gateway, Gibson, etc). The cloning...
  18. Open Enzyme Collection

    Type
    Collection
    ...include polymerase chain reaction (PCR), cloning, isothermal amplification, reverse transcription of RNA into...Enzyme Collection DNA Ligases DNA Polymerases Restriction Endonucleases Reverse Transcriptases RNA Polymerases...polymerases, DNA ligases, reverse transcriptases, and restriction enzymes. These enzymes are useful in both basic...for fixing damaged and degraded DNA prior to amplification, enzymes for cutting protein sequences to remove...the links below: DNA Ligases DNA Polymerases Restriction Endonucleases Reverse Transcriptases RNA Polymerases...-phi29pol phi29 DNA Polymerase Return to top Restriction Endonucleases Addgene ID Plasmid Gene/insert ...
  19. Plan Your Experiment

    Type
    Guide
    ...mixed cell population. The region of interest is PCR amplified, PCR products are denatured-renatured, treated...that introduce a novel restriction site. The region of interest is PCR amplified, digested with the appropriate...region of interest can be PCR amplified using primers that (A) flank the region of interest (deletions... The PCR product is then run on an agarose gel to determine whether the edit was successful. PCR amplification...appropriate restriction enzyme and run on an agarose gel to identify DNA fragments. PCR amplification and gel...an agarose gel to identify DNA fragments. PCR and restriction digest (for HDR repaired DSBs): For small...exact sequence of targeted alleles. Involves PCR amplification of targeted region from DNA, subcloning into...
  20. CRISPR Guide

    Type
    Collection
    ...enzymes to bind target DNA PCR P olymerase C hain R eaction; Used to amplify a specific sequence of DNA...experiments and must be amplified before use (Figure 8C), following the amplification protocol specified by...conditions: The sequence is unique compared to the rest of the genome The target is present immediately ...verify that the composition of your amplified library matches that of the original library. Next, you’... copies of the aptamer improves stability and amplifies the signal for better imaging of genomic loci,...Publications CRISPR Overview Bacteria have an interesting adaptive immune response that detects and eliminates... Cas enzyme and a gRNA specific to the gene of interest. The genomic target of the gRNA can be any ~20...
Showing: 41 - 60 of 70 results