...activity back to polymerase. In our case, dGTP will be used in the reaction, meaning that T4 Pol will remove...appropriate). The primer length is dependent on the T4 Pol "chew back" reaction, in which a single dNTP is included...guanosine residue and become stalled. A typical T4 Pol reaction is shown. Mix the components (add the T4...at room temperature for 30 minutes. Inactivate T4 Pol by heating to 75° for 20 minutes. Volume (μl) Reagent...will interfere with the exonuclease activity of T4 Pol in the following step. This may be accomplished by...Overhangs Treat your purified PCR product with T4 Pol in the presence of dCTP (not dGTP), corresponding...ligation. Because of its dual polymerase/exonuclease functions, T4 DNA polymerase can create overhangs of varying...

...resistant DNA polymerase from the thermophilic bacterium, Thermus aquaticus (Taq) . Taq polymerase can withstand...denature DNA polymerases from other species. In addition to the template DNA and the Taq polymerase, PCR requires... Taq polymerase has an optimal temperature around 70-75°C so this step enables the DNA polymerase to synthesize...elongation and synthesis. Taq DNA polymerase: Special DNA polymerase that can withstand radical temperature...Description of Polymerase Chain Reaction with protocol, tips and FAQ Protocols... Protocols Polymerase Chain Reaction...Reaction (PCR) Polymerase Chain Reaction (PCR) You may also like... Plasmid Cloning by PCR Restriction Digest...

... DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg/mL polybrene into 20 mL media. Thaw the...with DMEM complete + 10 µg/mL polybrene, the final concentration of polybrene in each well should be 10 µg...cells, resulting in a more homogenous (but still polyclonal) cell population. Depending on the transducibility...glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 ... the attachment of adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps...of the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just a sample of possible dilutions...Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene (µL) 0 0 500 1:5 300 200 1:10 150 350 1:50 30 ...

...μg/mL polybrene. TIP: Polybrene increases the efficiency of viral infection. However, polybrene is toxic...drives RNA Polymerase III transcription for generation of shRNA transcripts. cPPT Central polypurine tract,...especially repeated Ts because polyT is a termination signal for RNA polymerase III. Note that these were ... for 12-15 hours. c. In polypropylene microfuge tubes (do NOT use polystyrene tubes), make a cocktail .... Hexadimethrine Bromide (Polybrene) Prepare a 1mg/mL solution of polybrene (Sigma-Aldrich catalog #H9268...to adjust the sequencing conditions if the DNA polymerase has difficulty reading through the secondary ...Penicillin/Streptomycin Invitrogen: #15140-122 Polypropylene tubes VWR: #87003-290 Note: pLKO.1 could also...

...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...

...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...

...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...

...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...

...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:...

...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...

...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...

... 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...

... 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...

...264 50 mL conical tubes, VWR 89039-656 96-well polyester (clear) microplate seal, Thermo Scientific 5701...76322-134 Pipettes, 10 mL, VWR 89130-898 1 L polystyrene bottle, Corning 430518 PBS, 1X pH 7.4, VWR 45000...Desmin (blue) or a negative control protein, Apolipoprotein L3 (red) overnight, blocked, and incubated ...

...1814040 Microseal adhesive seal, Bio-Rad, MSB1001 Polystyrene Reservoirs, VWR, 89094-662 Microcentrifuge tubes... 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...

...a blunt end ligation. Use T4 DNA Polymerase or Klenow DNA Polymerase I for 3′ overhang removal and 5′ ...

...pipette Reagents 1X PBS Microcentrifuge tubes Sterile Poly-D-lysine coated coverslips HeLa cells 24-well plate...Procedure Section 1: Seeding cells Place a sterile poly-D-lysine coated coverslip in each well of a 24-well...

...cells with recombinant antibody plasmids using Polyethylenimine Max as a transfection reagent. After transfection... acid sodium salt, Sigma Aldrich P4543-10G Polyethylenimine hydrochloride, M.W. 40000 (PEI-MAX), Linear...

...Primers How to Design a Primer You may also like... Polymerase Chain Reaction Plasmid Cloning by PCR Agarose...

...anneal to each other. Phusion High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps...