...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette CO 2 incubator Pipet controller...96-well plate, seed one of the corner wells with ~1000 cells. This will help you focus the microscope later... incubating the cells. Pro-Tip Use the well with 1000 cells to focus the microscope, then you can easily...starting this procedure, be sure that the cell pool has been sufficiently selected with the appropriate antibiotic... antibiotic so that every cell in the culture has, in theory, been transduced. There are various schools...regularly for transgene expression. This protocol has been optimized for adherent cells but can be modified...

...mL conical collection tube. Centrifuge column at 1000 x g for 2 min to remove storage solution. Place ...container. Add 5 mL PBS to the column. Centrifuge at 1000 x g for 2 min to remove PBS. Repeat Steps 30–31 ...resin bed to fully absorb the sample. Centrifuge at 1000 x g for 2 min to collect the sample. Discard column...). Pro-Tip Add protease inhibitors to 1X if this has not already been done. We typically start with about... NanoDrop Spectrophotometer to see if the sample has reached the desired concentration. If the sample ... NanoDrop Spectrophotometer to see if the sample has reached the desired concentration. Gently transfer... Adjust pH to 7.0 Autoclave or filter sterilize 1000X protease inhibitor cocktail 25 mg leupeptin 50 mg...

...ensure that it has not drifted. Filter the solution through a 0.22 μm membrane. Aliquot 500–1000 μL into sterile...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Hemocytometer or cell ...system, Nalgene, 565-0010 (or for larger volumes 1000 mL 0.45 µm Rapid-Flow PES Filtration Unit, Nalgene... deionized water and adjust to a final volume of 1000 mL. Stir at RT until fully dissolved. Pro-Tip This...clogging during harvesting. The addition of sorbitol has been shown to increase viral titers by up to 1.8-...such as sorbitol and sodium bicarbonate, sorbitol has been found to increase viral titers by up to 1.8-...

...ensure that it has not drifted. Filter the solution through a 0.22 μm membrane. Aliquot 500–1000 μL into sterile...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...PEI such that the ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid...

...filter, VWR 431229 0.22 µm PES filtering system, 1000 mL, VWR 431098 0.45 µm PES filtering system, 500... aliquots and store at -20 °C until use. BCD TFX 1000 mL BalanCD HEK293 Media 10 mL 10% Pluronic-F68 40...final volume of 1 L and recheck pH to ensure that it has not drifted. Filter the solution through a 0.22 µm...incubate for 1 h at room temperature. After the BCD TFX has warmed in the bead bath, transfer the tubes to the...suggest preparing fresh solutions after one month. 1000X protease inhibitor cocktail 25 mg Leupeptin 50 mg...

...ensure that it has not drifted. Filter the solution through a 0.22 µm membrane. Aliquot 500–1000 µL into sterile...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 20–200 µL multichannel...Microcentrifuge tubes, Neptune 3745.X Pipette tips, 1000 µL, VWR 76322-154 Pipette tips, 10 µL, VWR 76322...periodically to determine when the desired color change has occurred. The color will change from yellow to blue...experimental conditions. When the desired color change has occurred, gently remove the plate seal. Using a multichannel...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette...calculate the titer for each sample: $$T = {R*C*D*1000\over V}$$ Where: T = Titer, GC/mL R = Reaction volume...sealer and press the ‘Seal’ button. After the plate has been sealed, proceed to thermocycling. Thermal Cycling...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...polyclonal cell population, meaning that the transgene has integrated in different locations in the various ...variety of dilutions and pick the population that has the most desirable level of expression. Over time...

...middle is hundreds, and the third is tens. Therefore, 1000 µL would read as 100 from top to bottom (as shown... flow rate into the pipette tip. Once the liquid has finished drawing into the pipette tip, lift the pipette...Anatomy of a Pipette This front view photo of a P1000 pipette shows the plunger at the top, which is used...tip off of the pipette. This side view photo of a P1000 pipette shows the volume adjustment ring right below...come in standard sizes: P2, P10, P20, P200, and P1000. The table shows the pipette with the appropriate...This image shows the P2, P10, P20, P100, P200, and P1000 as shown from left to right. Pipette Dispense Volume... 2 to 20 µL P100 10 to 100 µL P200 20 to 200 µL P1000 100 to 1,000 µL Each pipette should be used with...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette Ice bucket CO 2 incubator...mL) x dilution factor e.g., If the 1:100,000 well has 75 colonies, then there are 75 colonies /1.5 mL x...

...pipettes Ice bucket to hold antibiotic Antibiotic at 1000 x concentration disolved in the appropriate liquid...darken during the autoclave process if your sample has spent at least 10 min at 121 ℃. Use lab tape to label...antibiotic to the molten gel mix, you should create a 1000x stock solution. For example: If you’ll be preparing...

...MaxiPreps (Qiagen HiSpeed Max, Catalog #12663) Tips (1000 µL, 200 µL, 10 µL) Bacti Cell Spreaders (VWR, 60828... evenly with a sterile spreader until all liquid has been absorbed by the agar. This usually takes 1-2... 100 x 100 ÷ 0.1 This number should be at least 1000X greater than number of perturbations in library....least 10 colonies on the most diluted plate for 1000x coverage of a library of 100,000 plasmids. Colonies...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 2–50 µL multichannel pipette...sealer and press the ‘Seal’ button. After the plate has been sealed, proceed to thermocycling. Thermal Cycling...

...an impure AAV preparation. The 15% iodixanol step has 1M NaCl to destabilize ionic interactions between... and forth a few times to mix the iodixanol that has settled at the bottom of the column. We recommend...bring up the volume with formulation buffer. Use a P1000 to the bottom of the filter and pipette up/down ...

...antibiotic required for effective over-agar selection has been empirically determined. See selection curve ...without antibiotic High concentration (100 mg/mL, 1000x) carbenicillin stock solution in sterile water (...