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We narrowed to 852 results for: Met

Showing: 801 - 820 of 852 results

  1. Gibson Assembly Protocol

    Type
    Protocol
    ...effective method for the assembly of plasmids, and molecular biologists now use this method extensively...hundred kilobases. Nature Methods , 6(5), 343–345. https://doi.org/10.1038/nmeth.1318 (Link opens in a new...mitochondrial genome. Nature Methods , 7(11), 901–903. https://doi.org/10.1038/nmeth.1515 (Link opens in a new... Assembly Cloning You may also like... Cloning Methods Empty Backbone Guide Molecular Biology Protocols...the J. Craig Venter Institute developed a novel method for the easy assembly of multiple linear DNA fragments...intervening sequence between two PCR products, one method is to “stitch” together several oligos. This technique...

  3. Lentiviral Vector Guide

    Type
    Guide
    ...multiplexable method for genome-wide mapping of lentiviral integration sites. Molecular Therapy — Methods & Clinical...plasmid can be done with most standard cloning methods, including restriction enzyme, Gibson Assembly,...or may only be compatible with certain cloning methods (such as a Gateway destination vector), so be sure...your chosen plasmid is compatible with cloning methods available in your lab. When cloning your plasmids...HEK293T cells using your preferred transfection method. The HEK293T cells will produce the viral vectors...screens, lentiviral vectors are a preferred delivery method when conducting cellular barcoding experiments....lentiviral vectors, they have been seen as an effective method for in vivo gene delivery. Lentiviral vectors are...

  4. Adeno-associated virus (AAV) Guide

    Type
    Guide
    ...viral replication (E4, E2a and VA). These are sometimes combined within the packaging plasmid. For a summary...plasmid can be done with most standard cloning methods, including restriction enzyme, Gibson Assembly,...or may only be compatible with certain cloning methods (such as a Gateway destination vector), so be sure...your chosen plasmid is compatible with cloning methods available in your lab. When cloning your plasmids...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...Cas9 elements, making it one of the most common methods for in vivo CRISPR-based genome editing. Packaging...

  5. DNA Quantification

    Type
    Protocol
    ...purity. This can be done with a spectrophotometer. A spectrophotometer uses the absorbance/transmission... instructions for the spectrophotometer specific to your lab. Spectrophotometer Tips Before measuring ...labs have a NanoDrop, which is a very small spectrophotometer that can accurately read DNA concentration...measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution the DNA is resuspended in, ...NanoDrop. It is a good idea to re-zero any spectrophotometer between samples if they are expected to vary...

  6. Affinity Purification of Recombinant Antibodies with Protein A or Protein G

    Type
    Protocol
    ... we list the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and...compatible with 50 mL conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform...concentration of each fraction on the NanoDrop Spectrophotometer using the A280 IgG setting. Combine all eluates...concentration of the pooled sample on the NanoDrop Spectrophotometer using the A280 IgG setting. If the concentration...Determine antibody concentration on the NanoDrop Spectrophotometer. Dilute antibody to 1 mg/mL with PBS if needed...to check the concentration on the NanoDrop Spectrophotometer to see if the sample has reached the desired...Periodically check the concentration on the NanoDrop Spectrophotometer to see if the sample has reached the desired...

  7. Immunocytochemistry

    Type
    Protocol
    ...be the best fixation method for all. Alternative fixation methods such as methanol or acetone may be better... target protein using the formaldehyde fixation method. The protocol may need to be optimized for different... we list the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and.... Tips and Troubleshooting The optimal fixation method will vary depending on the sample type and the ...interest. You may need to try a variety of fixation methods to find the best conditions for your target. The...

  8. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...plasmid can be done with most standard cloning methods, including restriction enzyme, Gibson Assembly,...or may only be compatible with certain cloning methods (such as a Gateway destination vector), so be sure...your chosen plasmid is compatible with cloning methods available in your lab. When cloning your plasmids...cells (Figure 3A), similar to lentiviral packaging methods . The three plasmids described above (envelope,...these genes on separate plasmids ( in trans ). This method, therefore, reduces the number of plasmids that...pseudotyping. Figure 3: Gamma-retroviral vector packaging methods. Created with BioRender.com. Viral Vector Integration...protocol is for lentiviral vectors, but a similar method can be applied when using gamma-retroviral vectors...

  9. Adenovirus Guide

    Type
    Guide
    ...most popular choice due to their well-established methods, relative simplicity of production, and higher ...from the HV in final viral preparations. Several methods to avoid HV contamination have been developed, ...plasmid can be done with most standard cloning methods, including restriction enzyme, Gibson Assembly,...or may only be compatible with certain cloning methods (such as a Gateway destination vector), so be sure...your chosen plasmid is compatible with cloning methods available in your lab. When cloning your plasmids... by Bert Vogelstein’s lab , is the most popular method for creating first-generation rAdV vectors, which...supercoiled pAdEasy and the shuttle plasmid, but this method results in a higher background of non-recombinant...

  10. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...efficient guide (most guides would work, but they sometimes have different efficiency). And it is best if ...

  11. Modular Cloning Guide

    Type
    Guide
    ...functional plasmids. It is a powerful and very efficient method for creating many plasmids from different combinations...common set of components (Weber et al., 2011). This method, based on Golden Gate plasmid assembly, exploits...building synthetic genetic circuits, engineering metabolic pathways, assembling gene-editing tools, and more...editing, functional genomics, and more complex metabolic engineering and synthetic biology, compatible ...ubiquitination cascades of different organisms in E. coli . MetClo DNA Assembly Kit Bacterial Expression Christopher... for assembling large DNA fragments based on methylation-switching. 4G-Cloning Plasmid Kit Bacterial Expression...cellular circuits using Golden Gate-based cloning methods for use in mammalian cells. These can be used with...

  12. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...likely to retain stable transgene expression. Other methods of generating a monoclonal cell line include single-cell...controller Hazardous waste container Microscope Hemocytometer or other cell counter Reagents Polyclonal stable... growth of cells in individual cultures. Other methods for enhancing single cell growth, such as using...concentration in this homogenized cell solution using a hemocytometer or other cell counter. Pro-Tip Prepare a relatively...well plate. Sample Calculation First, use a hemocytometer to quantitate the homogenized cell solution....screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4. (Link opens in a new window...

  13. Fluorescence Titering Assay

    Type
    Protocol
    ...the dilution factors (method 1) or the volume of virus (method 2): Method 1 Method 2 $$T = {N*F*D\over ...with less than 40% fluorescent cells. Titering methods assume one integration event per cell. When the...

  14. Chemogenetics Guide

    Type
    Guide
    .... However, CNO is back-metabolized to clozapine and other clozapine metabolites and these can have off-target...concentrations of clozapine N ‐oxide (CNO). CNO is a metabolite of the antipsychotic clozapine and seems to be...experiments in this species may be entirely due to back-metabolism of CNO to clozapine, demonstrating the need for...activity, and do not seem to have the same back-metabolism issues as CNO and are attractive alternatives...2004). The highly efficacious actions of N-desmethylclozapine at muscarinic receptors are unique and not...

  15. Western Blot

    Type
    Protocol
    ...of the device. Select the desired method and make sure the parameters are correct. Transfer for 5–6 min... we list the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and...Pipette controller Pipette tips and pipettes Spectrophotometer Heat block Mini gel tank chamber Power supply...using a Pierce BCA Assay Kit or other preferred method for protein determination. Prepare a 50:1 Reagent...

  16. Lentivirus ddPCR Titration

    Type
    Protocol
    ...Titer by a Novel Droplet Digital PCR Method. Hum Gene Ther Methods . 2018 Apr;29(2):96-103. doi: 10.1089...Determine the concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples...reached, place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer...thermocycling. Thermal Cycling Run the following PCR parameters: Cycling Step Temperature (°C) Time (min) Ramp...

  18. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...is added to clearly distinguish the steps. This method will help enrich the prep with full (genome containing...adeno-associated virus purification using novel methods improves infectious titer and yield." Gene therapy...Wolfgang, and Lamla Thorsten. Human Gene Therapy Methods. August 2015, 26(4): 147-157. doi:10.1089/hgtb.2015.051...

  19. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...the first step toward efficient gene silencing. Methods for target selection are continuously being improved...and Transforming into Bacteria Use your ligation method of choice. For a standard T4 ligation, mix: 2 μL...Recommended Materials Material Vendor and catalog # Hexadimethrine bromide (polybrene)* Sigma-Aldrich: #H9268 ... et. al. Science . 1996 272:263-267. PubMed . Asymmetry in the assembly of the RNAi enzyme complex. . ...mL antibiotic. Pour plates and store at 4°C. Hexadimethrine Bromide (Polybrene) Prepare a 1mg/mL solution...

  20. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...Culture Introduction This protocol describes methodology for plating antibiotic over-agar for the selection...other small flame source) Incubator Reagents 6 cm diameter LB/agar plates without antibiotic High concentration...). It is, however, more expensive. With a 6 cm diameter petri plate containing solidified LB-agar, pipette...
Showing: 801 - 820 of 852 results