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  1. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the Video! Water Baths...plasmid using restriction enzymes Watch the Video! Polymerase Chain Reaction (PCR) Basic PCR protocol with ...Watch the Video! How to Design a Primer Key considerations when designing primers Watch the Video! Sequence...restriction digest and ligation Cloning by PCR Generate restriction sites by PCR Modification by Annealed...cloning with Type II restriction enzymes and T4 polymerase pLKO.1 - TRC Cloning Vector Cloning protocols...mouse genes. Virus Name Description Link to Video General Transfection Introduce plasmid DNA to mammalian...virus by counting antibiotic resistant colonies Generating Stable Cell Lines with Lentivirus Genomically...

  2. Kit Free RNA Extraction

    Type
    Protocol
    ...10, 2020 Equipment Refrigerated microcentrifuge If you only have a non-refrigerated microcentrifuge, see...extract RNA from tissues or cells without a kit. The general steps of this protocol include homogenization/lysis...X 10 7 cells. Allow sample(s) to sit at room temperature for 5 minutes to allow for dissociation of the...RNA degradation. If you don’t have access to a refrigerated centrifuge, you can carefully bring a centrifuge...centrifuge, bring this equipment back to room temperature, as prolonged storage in the cold room may damage...

  3. Centrifugation

    Type
    Protocol
    ... keep samples at specific temperatures. This protocol will cover the general procedure and features to...achieves this by using centrifugal force, which is generated by spinning the sample at a high speed. Being ...the bottom of a tube. You will likely encounter several different types of centrifuges that can accommodate...almost any centrifuge. Types and Parts There are several types of centrifuges: Mini or Tabletop Centrifuges... larger containers. Ultracentrifuges are also generally floor sized centrifuges that can hold larger containers...centrifuge settings according to speed and time (and temperature, if applicable) listed in your protocol. Pro-...

  4. Protocol - How to Design Primers

    Type
    Protocol
    ...capabilities. Taking into consideration the information above, primers should generally have the following properties...Primers How to Design a Primer You may also like... Polymerase Chain Reaction Plasmid Cloning by PCR Agarose... 5’ end of the primer usually has stretches of several nucleotides. Also, both of the 3’ ends of the hybridized...primers result in a slower hybridizing rate. On average, the DNA fragment that needs to be amplified should...content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer pairs should have a Tm...

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

    Type
    Protocol
    ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller Benchtop centrifuge compatible..., binding buffer, and elution buffer to room temperature (RT) before use. Reagent Preparation 1 M sodium... containing the recombinant antibody to room temperature (see Transfection for Recombinant Antibody protocol...30 kDa MWCO buffer exchange column. (Optional) Regenerate the column by washing with 25 mL of Protein A...remaining space in the column with PBS and pipette several times to mix. Centrifuge at 3100 x g for 8 min ...

  6. CRISPR Library Amplification

    Type
    Protocol
    ...amplification. This protocol is designed to be as general as possible but note that individual libraries ...our protocol on Bacterial Transformation . The generalized pooled library amplification protocol is expected... a diagnostic digest and high-throughput next generation sequencing (NGS). Select a restriction enzyme...10 colonies on the most diluted plate for 1000x coverage of a library of 100,000 plasmids. Colonies may... require extra incubation time in order to be enumerated accurately. Frequently the number of colonies...Ideally, dilutions would have been sufficient to enumerate single colonies but as long as one can ensure ...upon intended use. Typically yields range from several hundred micrograms to a milligram of total plasmid...

  7. Western Blot

    Type
    Protocol
    ...Calculate the average absorbance of the duplicate samples on the plate. Subtract the average absorbance ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...intended to support reproducibility in science. General Guidelines Acrylamide percentage varies in SDS-...depending on the size of your cell pellet but will generally be between 250–1000 µL . The ideal lysis buffer...TBST at RT on a shaking platform. Dilute the horseradish peroxidase-conjugated secondary antibody to the...

  8. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...L” shape, and then used like a cell spreader. Several other devices may be used for this purpose, provided...your petri plate. Incubate the plate at room temperature for at least 30 minutes with the lid on to give.../mL Carbenicillin plated over-agar Plate shows several individual colonies and effective selection. 150.../mL Carbenicillin plated over-agar Plate shows several individual colonies with smaller size than the ...plate). Please note we have found that there is generally a broad range of antibiotic concentrations that...

  9. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ... to room temperature. Now screw on the top of the bottle and store the LB at room temperature. When ready...bacteria since they will grow more slowly at lower temperatures. After incubation, check for growth, which is...plasmids should only need to be grown for 12-16 hr on average. Certain features of a plasmid may render it low...agar plate before growing in liquid culture. More aeration may help to increase the density of the culture...

  10. Lentiviral Vector Guide

    Type
    Guide
    ... into different generations. Lentiviral Generations First-generation First-generation lentiviral plasmids...Second-generation Third-generation Transfer Plasmid Can be packaged ONLY by a second-generation packaging...order to facilitate immunotherapy and gene therapy. Biomedicine & Pharmacotherapy , 128 , 110276. https...Figure 2: First-generation lentiviral plasmids. Created with BioRender.com. Second-generation Second-generation... All second-generation lentiviral transfer plasmids must be used with a second-generation packaging system...Figure 3: Second-generation lentiviral plasmids. Created with BioRender.com. Third-generation The third-generation...is often found in second-generation transfer plasmids as well. Third-generation plasmids include (Figure...

  11. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...reactions involve DNA fragments that have been generated by restriction enzyme digestion . Most restriction...depicts the ligation of two sticky ends that were generated by EcoRI digestion: Usually, scientists select...for details on optimization. Incubate at room temperature for 2hr, or at 16°C overnight (following the ...using "high concentration" ligase, 5min at room temperature is enough. For trickier ligations (such as ligation...cloning. While 3:1 will get you in the ballpark for average size genes and vectors, this ratio is really meant...

  12. Adenovirus Guide

    Type
    Guide
    ...potential applications for gene and cell-based therapies in the new era of personalized medicine . Genes & Diseases... table . Adenoviral Generations First-generation rAdV vectors The first generation of rAdV vectors was...formation is higher for first-generation rAdV vectors than later generations, which incorporate additional...Figure 2: First-generation rAdV vectors. Created with BioRender.com. Second-generation rAdV vectors Second-generation...Second-generation rAdV vectors contain the genomic deletions of first-generation rAdV vectors, as well...favor using first- and third-generation rAdV vectors. Figure 3: Second-generation rAdV vectors. Created with...with BioRender.com. Third-generation rAdV vectors Third-generation rAdV vectors are also referred to as...

  13. Promoters

    Type
    Guide
    ...the RNA polymerase. A transcription complex is constructed from the RNA polymerase and several transcription...because they control the binding of RNA polymerase to DNA. RNA polymerase transcribes DNA to mRNA which is ultimately... factors in order for RNA polymerase II (a eukaryote-specific RNA polymerase) to bind to a promoter. Transcription...the RNA polymerase binding site, TATA box, and transcription start site (TSS). RNA polymerase will bind... is not unusual to have several regulatory elements such as enhancers several kilobases away from the ...promoter, which is recognized by the RNA polymerase, an operator, a segment of DNA in which a repressor ...T7 RNA polymerase Promoter from T7 bacteriophage Sp6 Constitutive but requires Sp6 RNA polymerase Promoter...

  14. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...iodixanol step has 1M NaCl to destabilize ionic interactions between macromolecules. The 40% and 25% steps...prep with full (genome containing) particles. On average, iodixanol-purified AAV preparations contain ~20%...methods improves infectious titer and yield." Gene therapy 6.6 (1999): 973-985 (Link opens in a new window... Rist Wolfgang, and Lamla Thorsten. Human Gene Therapy Methods. August 2015, 26(4): 147-157. doi:10.1089...

  15. CRISPR Guide

    Type
    Guide
    ...using dual nickases to generate a knockout or gene edit; or using Cas9 to generate large genomic deletions...proteins. Large deletions generated by Cas3 have fewer off-target alterations, such as small mutations ...nucleases (TALENs) required scientists to design and generate a new nuclease pair for every genomic target. ..., and precisely edit DNA and RNA. The ease of generating gRNAs makes CRISPR one of the most scalable genome...and some of the ways the CRISPR components are leveraged to achieve specific goals, such as targeting multiple...depends on which Cas protein you use, and there are several to choose from. Many variations of Cas9 exist, ...will form a ribonucleoprotein complex through interactions between the gRNA scaffold and surface-exposed...

  16. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...tubes 96-well optical plate Pipette tips General Considerations Always run standards and samples in duplicate... master mix which contains a high-quality DNA polymerase and a blend of dTTP/dUTP to minimize carryover...Prepare a plasmid stock of 2 x 10 9 molecules/μL to generate a standard curve: One option is to use plasmid... be seen. The presence of a second peak at a temperature of ~70–75 °C usually indicates the presence of...

  17. Adeno-associated virus (AAV) Guide

    Type
    Guide
    ...ideal for gene therapy, neuroscience research, and other biomedical fields. Gene Therapy One of the major...low risk of insertional mutagenesis. Several AAV-based gene therapies have been approved by the US Food ...AAV) as novel vectors for human gene therapy . Current Gene Therapy, 3 (4), 281–304. https://doi.org/10.2174...serotypes: vector toolkit for human gene therapy . Molecular Therapy, 14 (3), 316–327. https://doi.org/10.1016... cycle. AAVs are therefore non-pathogenic and generally do not trigger a strong immune response. These...preferred choice to be used as viral vectors for gene therapy applications. This guide contains many viral vector-specific....com. Genome Integration Recombinant AAVs are generally considered to not integrate into the host genome...

  18. Antibody Guide

    Type
    Guide
    ...signaling molecules are horseradish peroxidase (HRP) and fluorophores. Horseradish peroxidase (HRP) HRP ...with several suitable antibodies; if they all meet your criteria, you may either purchase several to compare...epitopes on one antigen. Monoclonal antibodies are generated from cultured B cells, where each culture should...variability. Other Affinity Reagents There are several alternatives to antibodies, ranging from antibody... aliquots of at least 10 µL. Antibodies cannot tolerate freeze-thaw cycles. Here are a few tips for storing...a chemiluminescent reaction in the presence of several different substrates. The luminescence produced...Applications Antibody-based applications can be generally classed into four categories: Quantification -...

  19. Molecular Biology Reference

    Type
    Guide
    ...can be generated. The combination of a DNA fragment with a plasmid or vector DNA backbone generates a recombinant...DNA polymerase adding nucleotides. The major difference in this process occurs when the polymerase incorporates...referred to as cloned DNA, and this process of generating multiple identical copies of a recombinant DNA...Site (MCS) Short segment of DNA which contains several restriction enzyme sites, enabling easy insertion...expression will be driven by the promoter. As a general rule, the restriction sites in the MCS are unique...to work with - Plasmids are a convenient size (generally 1,000-20,000 basepairs) for physical isolation...gene. The promoter region is required for the generation of RNA from the insert DNA via transcription....

  20. Cloning

    Type
    Guide
    ... vectors that has a DNA topoisomerase I fused to the 3’ end. The topoisomerase acts as a ligase that joins...exonuclease, polymerase and ligase. 5' exonuclease digests the 5' end of dsDNA fragments to generate 3' single-stranded...fragments generating long overhangs that anneal to each other due to their homology. DNA polymerase then closes...years to move around pieces of DNA. Oftentimes several approaches will work for any specific cloning project...research project . The following guide will highlight several of the most popular cloning methods used to create...plasmids used for DNA cloning and expression contain several popular restriction enzyme sites. You can easily...TOPO (Image from Plasmid 101: TOPO Cloning ) Toposiomerase based cloning, often called TOPO® cloning or...
Showing: 181 - 200 of 1000 results