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We narrowed to 957 results for: omp

Showing: 121 - 140 of 957 results

  1. Lentiviral Vector Guide

    Type
    Guide
    ...laboratory, the non-required components have been removed, and the required components have been separated into...our lentiviral guide to learn about lentiviral components, generations, lentiviral production, and common...hence the use of “retro”. Lentiviruses have more complex genomes, containing packaging genes and accessory...require different isoforms of these packaging components. Therefore, lentivirus may not be efficiently...) necessary for integration. Not all of these components are necessary for lentiviral production in the...safety. For a summary of all lentiviral plasmid components, see the Lentiviral Plasmid Elements table . ...improvements and the potential for replication-competent lentivirus production — that is, creation of viral...

  2. CRISPR Library Amplification

    Type
    Protocol
    ... Thaw 4 tubes of electrocompetent cells on ice for 15-20 minutes or until completely thawed. Chill a box...obtained. Ensure that electrocompetent cells are being used. Chemically competent cells will not provide... to the inherent possibility of altering the composition of the library. Bottlenecks, fitness differences... in the morning to ensure that outgrowth and competition are limited. Equipment Table top centrifuge BioRad...MicroPulser TM , Bio-Rad 1652100) Reagents 200 µL electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen...Stbl4 cells or other ultra-high efficiency electrocompetent cells that are suitable for unstable or recombination-prone...recombination-prone DNA. The use of electrocompetent cells is essential to ensure high efficiency uptake...

  3. Molecular Cloning Techniques

    Type
    Guide
    ...overhang (blunt ends). Two pieces of DNA that have complementary overhangs, or which are both blunt-ended, can...This can make it difficult at times to identify compatible restriction enzymes that cut your insert or backbone... in the DNA sequence and can be time-consuming compared to other cloning methods. Read more in our Restriction... into a Gateway donor plasmid, which contains compatible attP sites via a proprietary BP clonase, creating...Addgene), it can be rapidly shuttled into any compatible Gateway destination vector, which contains attR...restriction cloning, once your project has been completed via Gateway cloning. Addgene's collection contains...method that relies on the hybridization of the complementary base pairs adenine (A) and thymine (T). TOPO...

  4. Adeno-associated virus (AAV) Guide

    Type
    Guide
    ...formation of replication-competent viruses. While AAV themselves are replication incompetent, there is a rare ... viral vector guide to learn about AAV vector components, production and common uses. Science...bilayer surrounding the viral particle and are only composed of a protein capsid and the viral genetic material...simplex virus to replicate in the host cell and complete the lytic cycle. AAVs are therefore non-pathogenic...packaging plasmid. For a summary of all AAV plasmid components, see the AAV Plasmid Elements table . Figure ...have limited restriction sites or may only be compatible with certain cloning methods (such as a Gateway... so be sure to confirm your chosen plasmid is compatible with cloning methods available in your lab. When...

  5. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...replication-competent retroviruses. For a summary of all gamma-retroviral plasmid components, see the Gamma-Retroviral...gamma-retroviral guide to learn about gamma-retroviral components, retroviral production, and common uses. Science...require different isoforms of these packaging components. Therefore, gamma-retroviruses may not be efficiently...production, and scientists have made changes to these components for increased safety when producing viral vectors...produce gamma-retroviral vectors, these required components have been separated into distinct plasmids for...have limited restriction sites or may only be compatible with certain cloning methods (such as a Gateway... so be sure to confirm your chosen plasmid is compatible with cloning methods available in your lab. When...

  6. Plan Your Experiment

    Type
    Guide
    ... process of interest. Do you want to: Generate complete and permanent loss of gene expression or function...you are ready to start designing the specific components (Cas enzyme and guide RNA) for your experiment...genetic manipulations require different CRISPR components. Selecting the genetic manipulation you plan ...plan to perform will help you narrow down which components you’ll need for a given experiment. In the table... will cover strategies for designing the gRNA component for each category. Genetic Manipulation Application...Requires a repair template Lower editing efficiency compared to NHEJ knockout Cut Nick Base Edit Generate specific...perfect world, your gRNA sequence would be an exact complement to your target sequence with no homologous sites...

  7. Lentivirus ddPCR Titration

    Type
    Protocol
    ...layout is complete, select 'Run' to begin the droplet reading. When the droplet reading is complete, export...biosafety level 2+. Please ensure that you are in compliance with your institution’s biosafety regulations...Human, Bio-Rad, 10031244 Reagent Preparation DMEM Complete: 500 mL DMEM, high glucose, pyruvate 55 mL heat...serum 5 mL glutaGRO 50 U/mL benzonase: 15 mL DMEM Complete 3 µL of 250 U/µL benzonase Procedure Transducing...the following dilution series of virus in DMEM complete: Prepare 7 microcentrifuge tubes with the appropriate...Leave one well untransduced (add 150 µL of DMEM complete instead of a viral dilution). Remember, this is...Gently add 1.5 mL of 50 U/mL Benzonase in DMEM complete to each well. Incubate at 37 °C for 30 min. Gently...

  8. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...want to use electro-competent cells instead of the more common chemically-competent cells. The number of...sufficient to give specificity and to also be compatible with a standard PCR reaction. Therefore, our...design is similar, but we need to use the reverse complement to get PCR amplification. We can start similarly...the ORF). We now need to generate the reverse-complement of this sequence so that we can successfully ...amplify the ORF. You can generate the reverse-complement using existing software (a quick internet search... one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you...according to the manufacturer’s instructions for your competent cells. For most standard cloning, you can transform...

  9. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...fragment of interest) into a compatibly digested vector backbone. This is accomplished by covalently connecting...are compatible, meaning that the overhanging base pairs on the vector and insert are complementary, the...is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells... sticky ends on either side of the vector are compatible with each other, the vector is much more likely... will allow you to verify that the vector was completely digested and if phosphatase treated, that the...Interpretation Uncut vector - Checks viability of competent cells and verifies the antibiotic resistance of... 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in the ballpark...

  10. Adenovirus Guide

    Type
    Guide
    ...vector guide to learn about adenoviral vector components, production and common uses....bilayer surrounding the viral particle and are only composed of a protein capsid and the viral genetic material...plasmid. For a summary of all adenoviral plasmid components, see the Adenoviral Plasmid Elements table . ...genome could lead to the production of replication-competent adenoviruses (RCAs) that would be able to infect...have limited restriction sites or may only be compatible with certain cloning methods (such as a Gateway... so be sure to confirm your chosen plasmid is compatible with cloning methods available in your lab. When... package larger cargo. CRISPR-Cas9 Delivery As compared to other viral vectors, rAdV vectors can provide...

  11. Molecular Biology Reference

    Type
    Guide
    ...editing is most commonly accomplished using CRISPR technology. CRISPR is composed of a DNA endonuclease ... proteins and reside on chromosomes, which are composed of DNA. These findings culminated in the central... encoded by DNA and RNA into proteins. DNA is comprised of four nucleotide bases: adenine, thymine, cytosine...enable the isolation and purification of cellular components, such as DNA, but in practice this isolation ...expression of an endogenous gene. This is frequently accomplished through expression of an shRNA targeting the... Invitrogen; New England BioLabs E. coli B F dcm ompT hsdS(rB mB) gal ccdB Survival Invitrogen F- mcrA...nucleotides according to the sequence of the complementary strand. Mimicking the DNA replication process...

  12. Colony Formation Titering Assay

    Type
    Protocol
    ...Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...colony formation assay. Prepare a batch of DMEM complete containing 10 μg/mL polybrene by diluting 20 μL...fold serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume...Lentivirus or Previous Dilution (μL) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus...follows: Dilute 7,000 cells into 9.45 mL of DMEM complete containing 10 μg/mL polybrene. Mix well by pipetting...to 8 h and the media can be replaced with DMEM complete. However, antibiotic-containing media should not...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. Incubate...

  13. Using a Light Microscope Protocol

    Type
    Protocol
    ...of a compound light microscope with the main components labeled: Figure 1: Diagram of a compound light...electron microscopes that allow us to see cellular components with science-fiction levels of detail. You can...common types that you're likely to encounter: the compound light microscope. As the name suggests, light ... make it through the sample because different components of the specimen will refract and reflect the ...) using dyes or stains that add color to some components of the sample but not others. The light that ...that allow you to capture images directly to a computer, but many microscopes do not and you may have ...

  14. Plasmid Cloning by Restriction Enzyme Digest (with Protocols)

    Type
    Protocol
    ...want to use electro-competent cells instead of the more common chemically-competent cells. The number of...version of your insert flanked by restriction sites compatible with the recipient plasmid's MCS. However, you... one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you...Follow the manufacturer’s instructions for your competent cells. For most standard cloning, you can transform...transform 1-2μl of your ligation reaction into competent cells such as DH5alpha or TOP10. If using much...getting colonies, you might want to use higher competency cells. Additionally, if your final product is...you used only one enzyme or used enzymes with compatible overhangs you will need to verify the orientation...

  15. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...kills all of the cells. Prepare a batch of DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg...a range of dilutions of the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just a sample...Dilution Volume of Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene (µL) 0 0 500 1:5 300 200 ...,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or...all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the final concentration ...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. This is...

  16. Promoters

    Type
    Guide
    ... including transcription and the transcription complex....of a gene is initiated. Promoters are a vital component of expression vectors because they control the...Promoter binding is very different in bacteria compared to eukaryotes. In bacteria, the core RNA polymerase...other hand, the process in eukaryotes is much more complex. Eukaryotes require a minimum of seven transcription... eukaryotic transcription, bacteria still have complex systems of gene regulation, like operons. Operons...Eukaryotic Promoters Eukaryotic promoters are much more complex and diverse than prokaryotic promoters and span... start site (TSS). Eukaryotic promoters are so complex in structure that the DNA tends to fold back on...

  17. AAV Production in HEK293 Cells

    Type
    Protocol
    ... mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...min. Neutralize trypsin by adding 10 mL of DMEM Complete. Pipet back and forth vigorously multiple times...-175 flasks. Adjust volume to 300 mL with DMEM complete media and mix. Seed all cells in 1 CS2. Return...CS2 to help detach the cells, add 200 mL of DMEM complete media, then transfer the cells into a sterile ...sterile bottle. Rinse the CS2 with 100 mL of DMEM complete medium and pool with the cells harvested in the previous... with PEI and allow for the addition of other components, such as sorbitol and sodium bicarbonate, sorbitol...Pipet back and forth to resuspend each pellet completely. Combine resuspended pellets and keep on ice....

  18. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ... 21-mer targets in your gene B.2 Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO...vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC for expression...sequence), while the EcoRI site is preserved. For a complete map of pLKO.1 containing the 1.9kb stuffer, visit... nuclear import of the vector’s preintegration complex in the transduced cells. hPGK Human phosphoglycerate...about off-target effects. B.2 Ordering Oligos Compatible with pLKO.1 To generate oligos for cloning into... shRNA sequence flanked by sequences that are compatible with the sticky ends of EcoRI and AgeI. Forward...M0202S T4 DNA ligase buffer NEB #B0202S DH5 alpha competent cells Invitrogen #18258-012 Qiaquick gel extraction...

  19. Lentivirus Production

    Type
    Protocol
    ... 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Friday: Plate...your insert of interest Reagent Preparation DMEM Complete (also known as D10): 10% v/v FBS and 4 mM L-alanyl-L-glutamine...Plate 8×10 5 cells in a T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use cells ...packaging cells at 3.8×10 6 cells per plate in DMEM Complete in 10 cm tissue culture plates. Incubate the cells...at RT. During the incubation, add 10 mL of DMEM Complete to a 15 mL conical and an appropriate amount of...the media. Replace the media with 10 mL of DMEM Complete or OptiPro SFM. Incubate the cells. Virus can ...

  20. Molecular Biology Protocol - Restriction Digest of Plasmid DNA

    Type
    Protocol
    ...works for both of your enzymes. Most companies will have a compatibility chart, such as the (Link opens in...enzymes that have compatible sticky ends. See (Link opens in a new window) NEB's compatible cohesive ends...ends chart . If you cannot find compatible sticky ends, you will need to fill in the overhangs and conduct...Master Mix" consisting of all of the reaction components except for the DNA. Aliquot your DNA into individual...
Showing: 121 - 140 of 957 results