...Description of Polymerase Chain Reaction with protocol, tips and FAQ... Protocols Polymerase Chain Reaction (PCR) Polymerase... Polymerase Chain Reaction (PCR) You may also like... Plasmid Cloning by PCR Restriction Digest of Plasmid...Ligation Background Information A standard Polymerase Chain Reaction (PCR) is an in vitro method that allows... video below to learn how to perform Polymerase Chain Reaction (PCR). Basic PCR Program Initial Denaturation...polymerase that can withstand radical temperature changes during a typical PCR. The DNA polymerase has an...

...1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...Insert tab, then Chart . This will add a blank chart to your spreadsheet. In the Chart Editor , select ...select the Chart Type drop down menu and select Scatter chart. Change the data range and select the cells where...1000 µL single channel pipette Pipette controller Microcentrifuge Electrophoresis chamber Power supply ...running buffer. Fill the chamber with 1X MOPS running buffer. Make sure that the chamber is sealed. Load 5 µL...and unplug the electrophoresis chamber. Remove the gel from the chamber. Use the metal spatula to gently...and ~25 kDa corresponding to the heavy chain (HC) and light chain (LC) proteins, respectively. There should...

...1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette 20–200 µL multichannel pipette Multichannel pipette reagent reservoirs Pipette...determine when the desired color change has occurred. The color will change from yellow to blue and generally... desired color change has occurred, gently remove the plate seal. Using a multichannel pipette, add 100... and set aside. Aspirate the wells and use a multichannel pipette to add 200 µL wash buffer to each well...tube and invert several times to mix. Using a multichannel pipette, add 200 µL of blocking buffer to each...

...10 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 1–10 ...10 µL multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel pipette Vortex, VWR, 10153...Use multichannel pipettes for the dilution series. For dilutions 1–5, use the 1–10 µL multichannel pipette...polystyrene reagent reservoir. Using a 20–200 µL multichannel pipette, carefully add the dilution buffer to...dilution scheme listed in step 13. Use a single channel 1–10 µL pipette to add 5 µL of each viral sample...slowly to avoid generating aerosols. Be sure to change tips each time. Dilution 1 (20X): 5 µL in 95 µL...to 5 µL. For dilutions 6–8, use the 20–200 µL multichannel pipette set to 50 µL. For mixing between dilutions...

...0.1–1 mL single channel pipette 0.5–10 µL single channel pipette 20–200 µL single channel pipette 2–20 µL...Option 2 with a buffer exchange/concentration using an Amicon 30 kDa MWCO buffer exchange column. (Optional... Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment Class II, Type A2 Biological Safety ... µL single channel pipette Reagents Aspirating pipette, VWR 414004-265 5 mL pipette, VWR 89130-896 10 ...above 1.0 mg/mL proceed to Option 1 with a buffer exchange using a Zeba Spin 7 kDa MWCO desalting column....the same recombinant antibody. Section 2: Buffer exchange Choose Option 1 or Option 2 based on the concentration...concentration of the pooled sample above. Option 1: Buffer exchange using a desalting column The 10 mL Zeba Spin Desalting...

...10 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 1–10 ...10 µL multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel pipette Reagents Universal...i.e master mix + water Whenever possible use a multichannel pipet to minimize pipetting error and variability...immediately before use. Use a reservoir and a multichannel pipette to dispense the master mix into the ...red were calculated using this plasmid, but will change if you use a different plasmid. Sample Calculation...with >50 uL if your well contains 100 uL) Use a multichannel pipette to load the standards and samples onto...

...10 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 2–50 ...50 µL multichannel pipette 20–200 µL multichannel pipette Ice bucket 96-well freezer blocks Vortex, VWR...appropriate amount of media volume listed in the chart below. For the first virus dilution, add 160 µL ...dilution. Repeat for all dilutions as indicated in the chart below. Dilution Factor Virus Volume to Dilute Media...cartridge into the cartridge holder. Using a 2–50 µL multichannel pipet, load 20 µL of the reaction mixtures into...polystyrene reagent reservoir. Using the 20–200 µL multichannel pipet, load 70 µL of droplet generation oil ...droplets have been generated, use a 20–200 µL multichannel pipette to collect 40 µL of droplets. Pro-Tip...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...fresh media containing selection reagent Day 3–14: Change media as needed Day 14–18: Expand and harvest stable...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Reagents... one suitable with your protocols. FBS can be purchased already heat-inactivated, or it can be inactivated...lentivirus, above) are dying. Perform regular media changes and monitor the growth of the cells. Pro-Tips Depending...culture, so it is important to do regular media changes and maintain optimal growth conditions for the ...absence of cell death, the cell media should be changed every 2–3 days to maintain the dose of antibiotic...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000.... Optional: perform a media exchange at ~18 h post transfection. Changing the media can reduce toxicity...protocol can be used to produce AAV from one Five Chambers Cell-Stack (CS5) (Link opens in a new window) ...1000 µL single channel pipette Hemocytometer or cell counter Ice bucket CO 2 incubator Pipet controller...find one suitable for your protocols. FBS can be purchased already heat-inactivated, or it can be inactivated... until fully dissolved. Pro-Tip This step is challenging due to the high viscosity of PEG. Stirring under...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...fresh media containing selection reagent Days 3–14: Change media as needed Days 14–18: Stain cells and count...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Reagents... one suitable with your protocols. FBS can be purchased already heat-inactivated, or it can be inactivated...presence of selective reagent for 12 days with media exchanges every 3–4 days. Colonies were then stained with...presence of selective reagent for 12 days with media exchanges every 3–4 days. After 12 days of selection, no...

...have different mechanisms to disperse liquids, this protocol is focused on single channel pipettes. These...consider using a multichannel pipette. Please watch Addgene’s protocol on multichannel pipetting to learn...ensures the accuracy of your experiment and any changes to the amount you are dispensing can negatively...adjustment ring right below the plunger. This ring changes the pipette volume. Below that is the tip ejector...without cross contamination as long as the tip is changed between samples. Tips can come loose in a bag, ...accurately and precisely is important since even small changes in the volume could affect your experiments. Beyond...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette Pipette controller Pipette tips and pipettes Spectrophotometer Heat block...block Mini gel tank chamber Power supply iBlot 2 Gel Transfer Device Roller Spatula Platform shaker Cold...to remove residual water. Load the gel into the chamber of the SDS-PAGE gel tank. Pro-Tip Different SDS-PAGE...500 mL with deionized water. Mix well. Fill the chamber of the SDS-PAGE gel tank with the 1X running buffer...

...sequences from step B.1 into the oligos below. Do not change the ends; these bases are important for cloning...light. Short wavelength UV light will increase the chance of damaging the DNA. Purify the DNA using a Qiaquick...CO 2 for 12-15 hours. Day 3: j. In the morning, change the media to remove the transfection reagent. Replace...to cells. Days 3+: e. Examine cells each day and change to fresh puromycin-containing media every other...Target cells should be approximately 70% confluent. Change to fresh culture media containing 8 μg/mL polybrene...Incubate cells at 37°C, 5% CO 2 overnight. Day 3: e. Change to fresh media 24 hours after infection. TIP: If...control for the puromycin selection. Days 4+: g. Change to fresh puromycin-containing media as needed every...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container pH ... one suitable with your protocols. FBS can be purchased already heat inactivated or it can be inactivated...panels: bright field images; right panels: GFP channel images. Last reviewed on: August 2, 2023...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Inverted... one suitable with your protocols. FBS can be purchased already heat-inactivated or it can be inactivated...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Reagents... one suitable with your protocols. FBS can be purchased already head inactivated or it can be inactivated...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette CO 2 incubator Pipet controller Hazardous waste container Microscope Hemocytometer... In the absence of selective medium, there is a chance that the integrated transgene could be lost through...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette Reagents 1X PBS Microcentrifuge tubes Sterile Poly-D-lysine coated coverslips...

... Nonetheless, we recommend performing a buffer exchange before using the purified AAV in vivo . The iodixanol... Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note: Both steps could be completed...Pool clean fractions. Concentration and buffer exchange: Stocks of Pluronic F68 Pluronic F68, 10% solution...Choi, and R. Jude Samulski. "Production and characterization of adeno-associated viral vectors," (Link ...

...restriction digest results. Equipment Electrophoresis chamber Pipetman Reagents Liquid DNA aliquot of your plasmid...enzymes. Most companies will have a compatibility chart, such as the (Link opens in a new window) double...in a new window) NEB's compatible cohesive ends chart . If you cannot find compatible sticky ends, you...