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We narrowed to 47 results for: TOR

Showing: 1 - 10 of 47 results

  1. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning vector. The vector is 8901 base...-Cloning Vector I.2 Recipes I.3 Warranty information Back to Top A. pLKO.1-TRC Cloning Vector A.1 The ...use of this vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the...TRC-cloning vector. Addgene Plasmid Description pLKO.1 – TRC control Negative control vector containing... Tools page. Several other laboratories have deposited pLKO derived vectors that may also be useful for...TRC Cloning Vector The following steps were the prevailing guidelines when the pLKO.1 vector was created...Digest pLKO.1 TRC-cloning vector with AgeI. Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA...

  2. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...plasmid Cut vector - Background due to uncut vector Cut vector + Background due to vector re-circularization...ends on either side of the vector are compatible with each other, the vector is much more likely to ligate...fuse the two ends of the vector together). Protocol: Standard Insert + Vector DNA Ligation Before setting...amount of cut insert and vector to use for the ligation reaction. The volume of vector DNA and insert DNA used...where the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will work just fine. We ...opens in a new window) ligation calculator to easily quantify how much vector and insert DNA to use. Combine... ALWAYS do a vector alone + ligase control. This will allow you to verify that the vector was completely...

  3. Plasmid Modification by Annealed Oligo Cloning (with Protocols)

    Type
    Protocol
    ...of this tutorial we will discuss how to add new restriction sites to the MCS of an empty vector. However...purify vector While waiting for your oligos to arrive, conduct a restriction digest of 1μg of vector with...Mix the annealed oligos with cut vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard...insert of 50bp in length into a 5kb vector, mix 100ng of the vector with 0.75-6 ng of annealed oligos)....just as easily to add short tags or shRNAs to any vector (the procedure will simply differ in terms of primer...primer design). Let's assume that your favorite vector has a relatively limited MCS (BamHI - EcoRI - SalI...restriction sites and add them to your existing vector. It is a couple days of work that will pay off ...

  4. Ligation Independent Cloning

    Type
    Protocol
    ...annealed but nicked vector product is then repaired during the replication cycle. Empty vectors for LIC typically...your PCR primers. Step 2: Linearize Vector In this example, the vector is cut at two points by the enzyme...subsequent reactions. Step 3: Create Vector Overhangs Treat the linearized vector with T4 DNA polymerase to "chew...treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing...allows for electrophoretic separation of linearized vector from the reaction mixture, and may provide counter-selection...shown here. The manufacturer of an LIC-specific vector will provide the homologous sequence which must...Search Addgene's collection of empty LIC cloning vectors Protocol Step 1: Design Your Primers Primer design...

  5. AAV Production in HEK293 Cells

    Type
    Protocol
    ...into sterile tubes. Store the tubes at -80 °C. After thawing, the solution can be stored at 4 °C for up to...before putting the CS5 in the incubator. Return the CS5 to the incubator for 96 h. This incubation time...Cells You may also like... AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference...Hemocytometer or cell counter Ice bucket CO 2 incubator Pipet controller Hazardous waste container pH ...pH meter Stir plate Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T cells (... glutaGRO 11 mL of 200 mM L-alanyl-L-glutamine. Store at 4 °C. D1 + 0.1 M sorbitol (optional) : DMEM ... solution may separate into phases. Aliquot and store at 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150...

  6. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ... PPE protocols . Working in a BSL-2 laboratory requires laboratory glasses in addition to coat and gloves...also adhered to. None of the authors, contributors, administrators, or anyone else associated with Addgene... eat, drink, chew gum, or apply makeup in the laboratory. Before you start your experiment, make sure ...Only mechanical pipetting should be done in the laboratory. Don’t mouth pipette! There may be some times...officer. BSL-2 Guidelines Remember, the BSL-1 laboratory guidelines above are expected to be followed ...not be worn outside of the BSL-2 area. BSL-2 laboratories must be clearly marked as “BSL-2.” The names...names and contact information of the laboratory manager should be clearly visible in the room and on the ...

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

    Type
    Protocol
    ...personal protective equipment including laboratory coat, laboratory goggles, and gloves. Sodium azide containing...Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller Benchtop centrifuge compatible...tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 °C bead... sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to 1 month at 4 °C Adjust pH to 7.0 Autoclave... Autoclave or filter sterilize 1000X protease inhibitor cocktail 25 mg leupeptin 50 mg antipain 250 mg...Recombinant Antibody protocol ). Pro-Tip Add protease inhibitors to 1X if this has not already been done. We typically...of supernatant and add 250 µL of 100X protease inhibitor cocktail. Add 1 part Protein A/G binding buffer...

  8. Protocol - How to Create a Bacterial Glycerol Stock

    Type
    Protocol
    ... are important for long-term storage of plasmids. Although you can store your plasmid DNA at -20°C, many...LB agar plate can be stored at 4°C for a few weeks. However, if you want to store bacteria for a longer...concentration of long-term glycerol storage is unknown. Most labs store bacteria in 15-25% glycerol. You ...for long-term storage. Protocols... Protocols Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids Creating...Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids You may also like... Bacterial Transformation...cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years. Protocol Video ...

  9. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...Equipment Biosafety Cabinet 4 °C Refrigerator Micropipettes and tips Pipettor and pipettes Benchtop centrifuge...conical tubes Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 °C bead...sterile 0.22 µm filter. Prepare 10 mL aliquots and store the solution at -20 °C. 1 mg/mL PEI-MAX Add 1 g ... through a 0.22 µm filter. Prepare aliquots and store at -20 °C until use. BCD TFX 1000 mL BalanCD HEK293...mL 200 mM Glutagro Do not add selective reagents Store at 4 °C until use. We suggest preparing fresh solutions...500 mL BalanCD HEK293 Feed 20 mL 200 mM Glutagro Store at 4 °C until use. We suggest preparing fresh solutions...solutions after one month. 1000X protease inhibitor cocktail 25 mg Leupeptin 50 mg Antipain 250 mg Benzamidine...

  10. Lentivirus ddPCR Titration

    Type
    Protocol
    ...transduce cells: Initial dilution factor of virus stock Dilution factor after additional 10-fold dilution...ddPCR) for lentivirus vector titration. Protocols... Protocols ddPCR Titration of Lentivirus Vectors ddPCR Titration of...of Lentivirus Vectors You may also like... Lentivirus Colony Formation Titration Assay Lentivirus Fluorescence...droplet digital PCR (ddPCR) to titer lentivirus vectors. This protocol specifically uses primers and probes...dilutions as indicated in the chart below. Dilution Factor Virus Volume to Dilute Media Volume Total Volume...fold dilution of the virus stock! Don’t forget to factor this into your calculations later (see calculation...
Showing: 1 - 10 of 47 results