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Addgene

Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

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We narrowed to 403 results for: Pol;

Showing: 341 - 360 of 403 results

  1. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    .... 0.1% Poloxamer 188 in PBS (A) Add 500 µL of 10% Poloxamer 188 to 49.5 mL PBS 0.01% Poloxamer 188 in ...Stocks of Poloxamer 188 Poloxamer 188, 10% solution A: 0.1% in PBS: 49.5 mL PBS + 500 µl Poloxamer 188 B:... Hamilton 1X PBS pH 7.4 1X PBS-MK buffer 100X Poloxamer 188 NaCl MgCl 2 KCl Centrifugal filter units (... (B) Add 5 mL of Buffer A to 45 mL PBS 0.001% Poloxamer 188 in PBS + 200 mM NaCl (C) (formulation buffer... Cover the filter membrane with 15 mL of 0.1% Poloxamer 188 PBS and incubate for 10 min at RT Spin at ... discard the flow through. Add 15 mL of 0.01% Poloxamer 188 PBS and incubate for 10 min at RT. Spin at...discard the flow through. Add 15 mL of 0.001% Poloxamer 188 + 200mM NaCl PBS. Spin at 3000 rpm for 5 min...

  2. Colony Formation Titering Assay

    Type
    Protocol
    ...complete containing 10 μg/mL polybrene by diluting 20 μL of 10 mg/mL polybrene into 20 mL of media. If using...glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 ... mL luer-lock syringe, BD BD300912 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps... before transduction. Note that in that case, polybrene should only be added at the time of viral transduction... collected virus, filter through a 0.45 μm polyethersulfone filter to remove cells and debris. Lentiviral...lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume of Lentivirus or Previous Dilution...) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus Added to Plate (μL) Volume...

  3. Colony Formation Titering Assay

    Type
    Protocol
    ...complete containing 10 μg/mL polybrene by diluting 20 μL of 10 mg/mL polybrene into 20 mL of media. If using...glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 ... mL luer-lock syringe, BD BD300912 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps... before transduction. Note that in that case, polybrene should only be added at the time of viral transduction... collected virus, filter through a 0.45 μm polyethersulfone filter to remove cells and debris. Lentiviral...lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume of Lentivirus or Previous Dilution...) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus Added to Plate (μL) Volume...

  4. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...how to generate a monoclonal cell line from a polyclonal pool of stable cells....how to generate a monoclonal cell line from a polyclonal pool of stable cells. Transducing cells with ...with lentivirus results in a heterogenous polyclonal population that varies in the number of integration ... as the lower expressing clones take over the polyclonal cell pool. Generating a monoclonal cell line ... Hemocytometer or other cell counter Reagents Polyclonal stable cell pool DMEM high glucose, Corning 10...Corning 3516 96-well plate, Corning 3596 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (optional) 40 µm...FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Polyclonal stable cell pool: see our protocol for generating...

  5. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...how to generate a monoclonal cell line from a polyclonal pool of stable cells....how to generate a monoclonal cell line from a polyclonal pool of stable cells. Transducing cells with ...with lentivirus results in a heterogenous polyclonal population that varies in the number of integration ... as the lower expressing clones take over the polyclonal cell pool. Generating a monoclonal cell line ... Hemocytometer or other cell counter Reagents Polyclonal stable cell pool DMEM high glucose, Corning 10...Corning 3516 96-well plate, Corning 3596 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (optional) 40 µm...FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Polyclonal stable cell pool: see our protocol for generating...

  6. Lentivirus Production

    Type
    Protocol
    ...vector transfected into 293T cells using a polyethylenimine (PEI) transfection protocol. This procedure... Thermo Fisher, 12309019 25 mM chloroquine Polyethylenimine, linear MW 25,000 Da Heat-inactivated FBS ... the attachment of adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 Microcentrifuge ...Hydrochloric acid Sodium hydroxide 0.22 μm polyethersulfone (PES) filter Transfection grade DNAs: Lentiviral...lab by heating to 56°C for 30 min. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI) Dissolve 100 mg...harvests, transfer the harvested media to a polypropylene storage tube and store at 4 °C between harvest...

  7. Lentivirus Production

    Type
    Protocol
    ...vector transfected into 293T cells using a polyethylenimine (PEI) transfection protocol. This procedure... Thermo Fisher, 12309019 25 mM chloroquine Polyethylenimine, linear MW 25,000 Da Heat-inactivated FBS ... the attachment of adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 Microcentrifuge ...Hydrochloric acid Sodium hydroxide 0.22 μm polyethersulfone (PES) filter Transfection grade DNAs: Lentiviral...lab by heating to 56°C for 30 min. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI) Dissolve 100 mg...harvests, transfer the harvested media to a polypropylene storage tube and store at 4 °C between harvest...

  8. AAV ddPCR Titration

    Type
    Protocol
    ...Supermix for Probes no dUTP, Bio-Rad,1863023 10% Poloxamer 188, Thermo Fisher, 24040032 Droplet generation...1814040 Microseal adhesive seal, Bio-Rad, MSB1001 Polystyrene Reservoirs, VWR, 89094-662 Microcentrifuge tubes...Reagent Preparation 1X PCR Buffer containing 0.05% Poloxamer 188: Prepare immediately before use and vortex...using. 500 µL of GeneAmp 10X Buffer 25 µL of 10% Poloxamer 188 4475 µL Molecular Biology Grade Water Procedure... the NTC. Pour the 1X dilution buffer into a polystyrene reagent reservoir. Using a 20–200 µL multichannel...cartridge. Add 800 µL of droplet generation oil to a polystyrene reagent reservoir. Using the 20–200 µL multichannel...

  9. AAV ddPCR Titration

    Type
    Protocol
    ...Supermix for Probes no dUTP, Bio-Rad,1863023 10% Poloxamer 188, Thermo Fisher, 24040032 Droplet generation...1814040 Microseal adhesive seal, Bio-Rad, MSB1001 Polystyrene Reservoirs, VWR, 89094-662 Microcentrifuge tubes...Reagent Preparation 1X PCR Buffer containing 0.05% Poloxamer 188: Prepare immediately before use and vortex...using. 500 µL of GeneAmp 10X Buffer 25 µL of 10% Poloxamer 188 4475 µL Molecular Biology Grade Water Procedure... the NTC. Pour the 1X dilution buffer into a polystyrene reagent reservoir. Using a 20–200 µL multichannel...cartridge. Add 800 µL of droplet generation oil to a polystyrene reagent reservoir. Using the 20–200 µL multichannel...

  10. Lentivirus ddPCR Titration

    Type
    Protocol
    ...EX0276-1 Benzonase 250 U/µl, Millipore #71205-3 Polybrene 10 mg/mL, Millipore, TR-1003-G Molecular Biology... Pierceable foil heat seal, Bio-Rad, 1814040 Polystyrene reservoirs, VWR, 89094-662 Microcentrifuge tubes...300,000 cells/well in 1350 µL media and 11.1 µg/mL Polybrene into the wells containing the virus and the untransduced...,000 cells in 13.5 mL of media and 11.1 µg/mL Polybrene. Mix the cell suspension well before seeding. ...final volume in the well is 1.5 mL, and the final Polybrene concentration is 10 µg/mL. Mix well and incubate...cartridge. Add 800 µL of droplet generation oil to a polystyrene reagent reservoir. Using the 20–200 µL multichannel...

  11. Lentivirus ddPCR Titration

    Type
    Protocol
    ...EX0276-1 Benzonase 250 U/µl, Millipore #71205-3 Polybrene 10 mg/mL, Millipore, TR-1003-G Molecular Biology... Pierceable foil heat seal, Bio-Rad, 1814040 Polystyrene reservoirs, VWR, 89094-662 Microcentrifuge tubes...300,000 cells/well in 1350 µL media and 11.1 µg/mL Polybrene into the wells containing the virus and the untransduced...,000 cells in 13.5 mL of media and 11.1 µg/mL Polybrene. Mix the cell suspension well before seeding. ...final volume in the well is 1.5 mL, and the final Polybrene concentration is 10 µg/mL. Mix well and incubate...cartridge. Add 800 µL of droplet generation oil to a polystyrene reagent reservoir. Using the 20–200 µL multichannel...

  12. Fluorescence Titering Assay

    Type
    Protocol
    ...glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without... the attachment of adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps... collected virus, filter through a 0.45 μm polyethersulfone filter to remove cells and debris. Lentiviral...lentivirus into DMEM complete containing 10 μg/mL polybrene. Note, this protocol was developed using low titer...Volume of DMEM complete (μL) Volume of 10mg/mL polybrene (μL) 1:10 150 1348.5 1.5 1:25 60 1438.5 1.5 1:...

  13. Fluorescence Titering Assay

    Type
    Protocol
    ...glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without... the attachment of adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps... collected virus, filter through a 0.45 μm polyethersulfone filter to remove cells and debris. Lentiviral...lentivirus into DMEM complete containing 10 μg/mL polybrene. Note, this protocol was developed using low titer...Volume of DMEM complete (μL) Volume of 10mg/mL polybrene (μL) 1:10 150 1348.5 1.5 1:25 60 1438.5 1.5 1:...

  14. AAV Production in HEK293 Cells

    Type
    Protocol
    ...by heating to 56 °C for 30 minutes. 0.45 μm polyethersulfone (PES) filter system, Nalgene, 565-0010 (or... the attachment of adherent cells) 1 mg/mL Polyethylenimine (PEI) 25 kDa MW Pro-Tip Other transfection...100 Benzonase/DNAse I (Millipore 71205-3) 40% Polyethylene Glycol 8000 (PEG) + 0.5 M NaCl Cell lysis buffer...HEPES to 750 mL DMEM + 1 g/L glucose. 1 mg/mL polyethylenimine (PEI) solution: Dissolve 100 mg of PEI powder...discard the tube and thaw a new working stock. 40% POLYETHYLENE GLYCOL (PEG) 8000 solution: Dissolve 400 g of...

  15. AAV Production in HEK293 Cells

    Type
    Protocol
    ...by heating to 56 °C for 30 minutes. 0.45 μm polyethersulfone (PES) filter system, Nalgene, 565-0010 (or... the attachment of adherent cells) 1 mg/mL Polyethylenimine (PEI) 25 kDa MW Pro-Tip Other transfection...100 Benzonase/DNAse I (Millipore 71205-3) 40% Polyethylene Glycol 8000 (PEG) + 0.5 M NaCl Cell lysis buffer...HEPES to 750 mL DMEM + 1 g/L glucose. 1 mg/mL polyethylenimine (PEI) solution: Dissolve 100 mg of PEI powder...discard the tube and thaw a new working stock. 40% POLYETHYLENE GLYCOL (PEG) 8000 solution: Dissolve 400 g of...

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

    Type
    Protocol
    ...plasmid using restriction enzymes Watch the Video! Polymerase Chain Reaction (PCR) Basic PCR protocol with ...cloning with Type II restriction enzymes and T4 polymerase pLKO.1 - TRC Cloning Vector Cloning protocols...Lentivirus Production Produce lentivirus with a polyethyenimine (PEI) transfection protocol Fluorescence Titering...Dilution Generate monoclonal cell lines from a polyclonal pool of stable cells AAV Production in HEK293...

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

    Type
    Protocol
    ...plasmid using restriction enzymes Watch the Video! Polymerase Chain Reaction (PCR) Basic PCR protocol with ...cloning with Type II restriction enzymes and T4 polymerase pLKO.1 - TRC Cloning Vector Cloning protocols...Lentivirus Production Produce lentivirus with a polyethyenimine (PEI) transfection protocol Fluorescence Titering...Dilution Generate monoclonal cell lines from a polyclonal pool of stable cells AAV Production in HEK293...

  18. General Transfection

    Type
    Protocol
    ... transfecting mammalian cells using linear polyethylenimine. Transfections allow for transient expression... Thermo Fisher, 12309019 25 mM chloroquine Polyethylenimine, linear MW 25,000 Da Microcentrifuge tubes...Hydrochloric acid Sodium hydroxide 0.22 μm polyethersulfone (PES) filter Syringes for filtering Reagent...should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI) Dissolve 100 mg...

  19. General Transfection

    Type
    Protocol
    ... transfecting mammalian cells using linear polyethylenimine. Transfections allow for transient expression... Thermo Fisher, 12309019 25 mM chloroquine Polyethylenimine, linear MW 25,000 Da Microcentrifuge tubes...Hydrochloric acid Sodium hydroxide 0.22 μm polyethersulfone (PES) filter Syringes for filtering Reagent...should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI) Dissolve 100 mg...

  20. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ... high fidelity taq polymerase to minimize mutations. The fidelity of the polymerase becomes more important...bp depending on the polymerase used. Because of this, no matter which taq polymerase you use, it is important...
Showing: 341 - 360 of 403 results