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We narrowed to 976 results for: nin

Showing: 941 - 960 of 976 results

  1. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...requires free nucleotides [dNTPs; adenine (A), cytosine (C), guanine (G), thymine (T)] in an equal molar...our protocol on plasmid cloning by PCR to learn how to design primers for cloning purposes. What do I do...Chain Reaction (PCR) You may also like... Plasmid Cloning by PCR Restriction Digest of Plasmid DNA DNA Ligation...DNA synthesis. Sterile dH 2 O: Used to fill the remaining 50 μL of the reaction mix. Its solvent and buffer...

  2. Protocol - How to Purify DNA from an Agarose Gel

    Type
    Protocol
    ...technique for molecular cloning, such as PCR - or restriction enzyme -based cloning. Protocol: Gel Purification... can be achieved by using a wider gel comb and running the gel at a lower voltage. You will want to have...resolution (crispness) of your DNA bands include: a) running the gel at a lower voltage for a longer period ...bands? If you have similarly sized bands that are running too close together you can adjust the gel percentage...

  3. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...DNA by centrifugation, and remove the plasmid-containing supernatant. Add either ethanol or isopropanol...and purity will vary. For more information on determining DNA concentration and purity click here . Top...overnight cultures from single colonies of bacteria containing your plasmid of interest. Add 1.5 mL of the stock... . Note: Phenol-chloroform extraction removes remaining contaminant proteins and RNase A from the DNA ...When phenol is mixed with the aqueous solution containing DNA, proteins will move into the phenol phase...

  4. Lentivirus ddPCR Titration

    Type
    Protocol
    ...such as glutaGRO, Corning 25-015-CI) 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations... pipettes and surfaces with 10% bleach. Safety Warnings Lentivirus is generally considered biosafety level..., VWR, 29442-042 DMEM, high glucose, pyruvate, Corning, 10-013-CV Heat-inactivated FBS L-alanyl-L-glutamine...media and 11.1 µg/mL Polybrene into the wells containing the virus and the untransduced control. Pro-Tip...

  5. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ...excise some or all of an insert from it. Before beginning your diagnostic digest, you will need to select...performing either PCR - or restriction enzyme -based cloning to test individual clones before use of more expensive...digested with restriction enzymes common to multiple cloning sites, will result in similar sized bands, thus...restriction digest. Although this is never an ideal cloning strategy, sometimes it cannot be avoided. If you...

  6. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...Ligation DNA Ligation You may also like... Plasmid Cloning by Restriction Enzyme Digest Restriction Digest...each other. When the sticky ends are compatible, meaning that the overhanging base pairs on the vector and...their concentration. However, for most standard cloning (where the insert is smaller than the vector) a... is not working or when doing more complicated cloning. While 3:1 will get you in the ballpark for average...

  7. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    .... The 60% step serves as a cushion for genome-containing virions. Phenol red is added to clearly distinguish...method will help enrich the prep with full (genome containing) particles. On average, iodixanol-purified AAV... first microcentrifuge tube under the needle’s opening to collect the fractions. Puncture the top of the...soon as the top of the tube is punctured, AAV-containing Iodixanol solution will flow from the needle ...

  8. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...Determine the number of genome-containing particles in an AAV prep using qPCR, SYBR Green Technology,...can be used to determine the number of genome-containing particles of an AAV prep using SYBR green technology...GGAACCCCTAGTGATGGAGTT rev ITR primer, 5'-CGGCCTCAGTGAGCGA ITR-containing plasmid for standard curve RNase-free DNase 10X.... When developing the assay multiple plasmids containing ITR were tested. Plasmid #59462 is one plasmid...

  9. Video Library

    Type
    Protocol
    ...technique for molecular cloning, such as PCR- or restriction enzyme-based cloning. Purifying DNA from an...equipment, setting up a clean workspace, and maintaining sterility while working. AAV Titration by qPCR...

  10. Centrifugation

    Type
    Protocol
    ...using centrifugal force, which is generated by spinning the sample at a high speed. Being able to separate...which you need to spin your samples. For example, spinning down samples in 1.5 mL microfuge tubes at 10,000...and investigate the issue. When you are finished spinning your samples, carefully remove them from the centrifuge...

  11. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ... that you have received hazardous waste safety training before starting the work. Before working with ...Biohazard signs should be placed in all areas containing biohazardous materials, including biohazardous... safety protocols require bloodborne pathogens training. It is strongly recommended that anyone participating...

  12. Molecular Biology Reference

    Type
    Guide
    ...Addgene Resource(s) Cloning Plasmids Used to facilitate the cloning of DNA fragments. Cloning vectors tend to...comprised of four nucleotide bases: adenine, thymine, cytosine, and guanine (abbreviated to A, T, C, and G,.... With current cloning technology, it is easy to create and modify plasmids containing the genetic element...plasmid-containing bacteria by providing a survival advantage to the bacterial host. Multiple Cloning Site...optimized to help in the initial cloning of a DNA fragment. Commonly used cloning vectors include Gateway entry...entry vectors and TOPO cloning vectors. Empty Backbones , Cloning Kits Expression Plasmids Used for gene...molecule is known as DNA or molecular cloning. The advent of molecular cloning enabled scientists to break chromosomes...

  13. CRISPR Guide

    Type
    Guide
    .... P., White, M. F., Yakunin, A. F., Garrett, R. A., Van Der Oost, J., . . . Koonin, E. V. (2015). An updated...in your lab, from knockouts to pooled library screening. Science...CRISPR Basics Small Edits Large Edits Genome-Wide Screening Cas9 Fusion Tools Cas9 Alternatives Resources ...the efficient but error-prone non-homologous end joining (NHEJ) pathway and the less efficient but high-... , and increases the likelihood that any cell containing the CRISPR plasmid will have all desired genomic...researchers to target anywhere from 2–7 genetic loci by cloning multiple gRNAs into a single plasmid — though some...coding sequence ~1 kb shorter than SpCas9 while retaining the same basic function. Its size allows it to...

  14. Adenovirus Guide

    Type
    Guide
    ...BioRender.com. Adenoviral Vector Production Cloning Cloning your transgene, gRNA, or shRNA of interest ...plasmid is compatible with cloning methods available in your lab. When cloning your plasmids, be sure to... coli cells containing pAdEasy. pAdEasy is a ~33 kb adenoviral backbone plasmid containing the adenoviral...shuttle plasmid containing the transgene of interest, and the adenoviral plasmid containing the adenoviral...(AdV) are medium-sized, non-enveloped viruses containing a linear double-stranded DNA (dsDNA) genome. ...Shuttle plasmid (also known as transfer plasmid) — containing the transgene of interest, the ITRs, and homology... the adenoviral plasmid. Adenoviral plasmid — containing the rest of the adenoviral genome necessary for...

  15. Plan Your Experiment

    Type
    Guide
    ...experiment, including selecting the edit type, designing and cloning a gRNA, delivering Cas and the gRNA, and... are a few factors to keep in mind when designing and cloning your gRNAs. Select Genetic Element to be... into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the ...your experimental goal, you are ready to start designing the specific components (Cas enzyme and guide ...the next section, we will cover strategies for designing the gRNA component for each category. Genetic ... cells will use error-prone non-homologous end joining (NHEJ) at the cut site Cas9 or Cas9 nickase High-fidelity... comes down to location, location, location. Positioning your chosen Cas enzyme at the right spot within...

  16. Lentiviral Vector Guide

    Type
    Guide
    ...lentiviral plasmids. Lentiviral Vector Production Cloning Cloning your transgene, gRNA, or shRNA of interest ...plasmid is compatible with cloning methods available in your lab. When cloning your plasmids, be sure to...retro”. Lentiviruses have more complex genomes, containing packaging genes and accessory genes specific ...transfer plasmid can be done with most standard cloning methods, including restriction enzyme, Gibson Assembly...restriction sites or may only be compatible with certain cloning methods (such as a Gateway destination vector),...However, if the plasmid contains a Gateway cassette containing the ccdB gene, a ccdB-resistant strain is necessary...necessary. For more information on cloning and working with plasmids, visit Addgene’s Molecular Biology...

  17. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...BioRender.com. Gamma-Retroviral Vector Production Cloning Cloning your transgene, gRNA, or shRNA of interest ...plasmid is compatible with cloning methods available in your lab. When cloning your plasmids, be sure to...of “retro”. Retroviruses have simpler genomes, containing only the necessary packaging genes, while lentiviruses...transfer plasmid can be done with most standard cloning methods, including restriction enzyme, Gibson Assembly...restriction sites or may only be compatible with certain cloning methods (such as a Gateway destination vector),...However, if the plasmid contains a Gateway cassette containing the ccdB gene, a ccdB-resistant strain is necessary...necessary. For more information on cloning and working with plasmids, visit Addgene’s Molecular Biology...

  18. Adeno-associated virus (AAV) Guide

    Type
    Guide
    ... with BioRender.com. AAV Vector Production Cloning Cloning your transgene, gRNA, or shRNA of interest ...plasmid is compatible with cloning methods available in your lab. When cloning your plasmids, be sure to...Viruses (AAV) are small, non-enveloped viruses containing a small, linear single-stranded DNA (ssDNA) genome...Transfer plasmid (also known as cis plasmid) — containing the transgene of interest between the two ITRs...Packaging plasmid (also known as Rep/Cap plasmid) — containing the Rep and Cap regions necessary for the production...and assembly of viral capsids. Helper plasmid — containing adenoviral genes not present in the AAV genome...transfer plasmid can be done with most standard cloning methods, including restriction enzyme, Gibson Assembly...

  19. Guide to Using Pooled Libraries

    Type
    Guide
    ...represent a powerful tool for forward genetic screening, or identifying previously unknown genes that ...the Moffat lab has over 175,000 different gRNA-containing plasmids). Amplifying and Using the Library Once...and resistance to to the nucleotide analog 6-thioguanine . Next-generation sequencing is the only way ...that you have access to this technology if you’re planning to conduct a pooled library screen. Figure 2: ...pass selection (in the case of a reporter) Sort “winning” cells Sequence those cells that pass (“win”) Get...CRISPR guide for more information on genome-wide screening with CRISPR/Cas9 . Browse our Viral Vector resources...some of our most popular libraries! Genome-wide Screening Using CRISPR/Cas9 Lentiviral CRISPR Libraries ...

  20. Immunocytochemistry

    Type
    Protocol
    ...protocol describes the basic steps for fixing and staining cells in culture with a primary antibody against...optimal dose. To ensure that your antibody is both functioning as expected and specific, include a positive ...
Showing: 941 - 960 of 976 results