...single channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel...multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel pipette Vortex, VWR, 10153-688 Mini...and pipette 5–10 times to mix. Dilute the virus as follows: Mix dilutions thoroughly, but pipette slowly...multichannel pipettes for the dilution series. For dilutions 1–5, use the 1–10 µL multichannel pipette set to...multichannel pipette set to 50 µL. For mixing between dilutions, use the 20–200 µL multichannel pipette set to...primers, and probe on ice before use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings ...droplet generation are supplied with sufficient pipette tips and reagent reservoirs. Pro-Tip Prepare the...

...single channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 2–50 µL multichannel...diluent, pipette 10 times, to remove virus from pipette tip. To mix the dilution, set the P200 pipette to 200...multichannel pipette 20–200 µL multichannel pipette Ice bucket 96-well freezer blocks Vortex, VWR, 10153-... 200 µL and pipette 10–20 times. Use a new pipette tip to transfer 200 µL of the first dilution into the...mixes and samples on ice before use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings ...well before seeding. Mix each well with a 1 mL pipette 5–10 times. The final volume in the well is 1.5...6-well plates. Be sure to use a new aspirating pipette for each well as to not contaminate cell conditions...

...channel pipette 20–200 µL multichannel pipette Multichannel pipette reagent reservoirs Pipette controller...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...Neptune 3745.X Pipette tips, 1000 µL, VWR 76322-154 Pipette tips, 10 µL, VWR 76322-132 Pipette tips, 200 µL... µL, VWR 76322-150 Pipette tips, 20 µL, VWR 76322-134 Pipettes, 10 mL, VWR 89130-898 1 L polystyrene bottle...microfuge tube and vortex. 0 ng/µL : 450 µL PBS only. Pipette 50 µL into the first three wells of each row, using...wash buffer (0.05% Tween-20 in PBS) as follows: Pipette 0.5 mL of Tween-20 to into 999.5 mL 1X PBS Cap ...aside. Aspirate the wells and use a multichannel pipette to add 200 µL wash buffer to each well. Washing...

...are commonly referred to as 'competent cells.' Many companies sell competent cells, which come frozen and...instructions that came with your competent cells. Pro-Tips Commercial competent cells range significantly in... LB or SOC media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80... To save money, many labs also make their own competent cells. This is a relatively simple procedure and...DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY...than for an intact control plasmid. Incubate the competent cell/DNA mixture on ice for 20-30 mins. Heat shock...usually ideal, but this varies depending on the competent cells you are using). Put the tubes back on ice...

...Heating plate Pipettors 1–10 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel...multichannel pipet to minimize pipetting error and variability Mix samples very well by pipetting back and...multichannel pipette Reagents Universal SYBR Master Mix 2X Primer pair targeting AAV2 ITR ( Aurnhammer et al., ...water Microcentrifuge tubes 96-well optical plate Pipette tips General Considerations Always run standards...that result from a variety of causes, such as pipetting error and sample evaporation. Make the master ...before use. Use a reservoir and a multichannel pipette to dispense the master mix into the wells. Reagent...

...obtained. Ensure that electrocompetent cells are being used. Chemically competent cells will not provide... 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...efficiency (1x10 10 cfu/µg) commercially prepared electrocompetent cells is strongly recommended. 800 ng pooled... library DNA (100 ng plasmid DNA per 25 µL electrocompetent cells) Less library DNA can be used but this...

...channel pipette Reagents Aspirating pipette, VWR 414004-265 5 mL pipette, VWR 89130-896 10 mL pipette, VWR...single channel pipette 0.5–10 µL single channel pipette 20–200 µL single channel pipette 2–20 µL single...VWR 89130-898 25 mL pipette, VWR 89130-900 50 mL pipette, VWR 89130-902 50 mL conical tubes, VWR 89039-...A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller Benchtop centrifuge compatible with ...tubes, VWR 76289-498 Before Starting Wipe down all pipettes and equipment with 10% bleach prior to use. Warm...the remaining space in the column with PBS and pipette several times to mix. Centrifuge at 3100 x g for...

...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...Invitrogen 26619 Pipette tips, 10 µL, VWR 76322-132 Pipette tips, 20 µL, VWR 76322-134 Pipette tips, 200 µL...VWR 76322-150 Pipette tips, 1000 µL, VWR 76322-154 Pipettes, 5 mL, VWR 89130-896 Pipettes, 10 mL, VWR 89130...single channel pipette Pipette controller Microcentrifuge Electrophoresis chamber Power supply Rocking ... 89130-898 Pipettes, 25 mL, VWR 89130-900 Pipettes, 50 mL, VWR 89130-902 Gel loading tips, Corning 4853...

...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...single channel pipette Hemocytometer or cell counter Ice bucket CO 2 incubator Pipet controller Hazardous...Neutralize trypsin by adding 10 mL of DMEM Complete. Pipet back and forth vigorously multiple times to obtain...with the cells harvested in the previous step. Pipette back and forth or shake vigorously to obtain a ...total of 5 mL of cell lysis buffer (recipe above). Pipet back and forth to resuspend each pellet completely...adding a total of 12.5 mL of cell lysis buffer. Pipet back and forth to resuspend each pellet completely...

...spread antibiotic over-agar. Equipment Pipette tips for both pipetting and spreading Bunsen burner (or other...expensive. With a 6 cm diameter petri plate containing solidified LB-agar, pipette 150 µL of carbenicillin on...liquid remaining on the surface). We use the micropipette tip itself to do the spreading; the tip is gently...used for this purpose, provided that they fit your petri plate. Incubate the plate at room temperature for...same way as the antibiotic, using either a bent micropipette tip or other cell spreading device that fits...

...Fix and label cells Equipment Pipette controller Pipette tips and pipettes Rocking platform Tweezers Fluorescent...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...single channel pipette Reagents 1X PBS Microcentrifuge tubes Sterile Poly-D-lysine coated coverslips HeLa...

...single channel pipette Pipette controller Pipette tips and pipettes Spectrophotometer Heat block Mini gel...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...

...be worn at all times. Use plastic pipettes in place of glass pipettes or needles. Liquid waste should be...vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC for expression...M0202S T4 DNA ligase buffer NEB #B0202S DH5 alpha competent cells Invitrogen #18258-012 Qiaquick gel extraction...hours. Transform 2 μL of ligation mix into 25 μL competent DH5 alpha cells, following manufacturer’s protocol...OPTI-MEM In a polypropylene tube, add OPTI-MEM first. Pipette FuGENE® directly into the OPTI-MEM – do not allow...tube from step c for a total volume of 100 μL. Pipette master mix directly into the liquid and not onto...cells. Swirl to disperse mixture evenly. Do not pipette or swirl too vigorously, as you do not want to ...

...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Inverted fluorescence...cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2 mL of cell suspension ...

...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container pH meter Stir...out of the previously seeded 10 cm plate. Slowly pipette the transfection mix onto the 10 cm plate, being...

...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Reagents DMEM...complete containing 10 μg/mL polybrene. Mix well by pipetting or inverting. Aliquot 1.35 mL of the cell suspension...

...Biosafety Cabinet 4 °C Refrigerator Micropipettes and tips Pipettor and pipettes Benchtop centrifuge compatible...Reagent Preparation instructions. Wipe down all pipettes and equipment with 10% bleach prior to use. Reagent...Check the cell density and viability: Using a 5 mL pipette, transfer 0.5 mL of HEK293 cell suspension into...the microfuge tube containing the trypan blue. Pipette 10 times to mix. Load 10 µL of the cell suspension...

...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Reagents DMEM...DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or inverting the tube. Aliquot 1 mL of cell suspension...

...single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000 µL single...single channel pipette CO 2 incubator Pipet controller Hazardous waste container Microscope Hemocytometer... by passing several times through a serological pipet or by passing through a 40 µm cell strainer mesh...

...storage in the cold room may damage it. Using a pipettor, transfer the top, aqueous phase to a new RNAse-free...disturb or collect the interphase layer with your pipette tip. Pro-Tip To collect as much of the top aqueous...interphase layer, consider using a lower volume pipettor like a p200 to collect a majority of the aqueous...