...the Qiagen HiSpeed Maxi Kit (one conical is its own Maxiprep). Critical Do not freeze pellets for later...libraries up to 200,000 individual plasmids and can be scaled according to the actual number. Required Quality...selection). This recombination, at a low rate, is not typically a problem as these sequences are not efficiently...200,000 individual plasmids. For larger libraries scale the number of cells and DNA accordingly. Pro-Tip...cells) Less library DNA can be used but this dramatically increases the chances of individual plasmids...Cell Spreaders (VWR, 60828-680) 5 mL and 10 mL Serological pipettes Ice slurry (Ice bucket with ice and ...normal growth is expected to work. 50 mL Falcon Conical tubes (Fisher, 14-432-22) Reagent Preparation Prepare...

... forget to factor this into your calculations later (see calculation example below). Seed 300,000 cells... a CSV file which will be used to calculate the titer. Calculations In the experimental setup above, the...20 200 40 400 80 800 160 1600 To calculate the titers, first calculate the number of viruses per cellular...) to titer lentivirus vectors. This protocol specifically uses primers and probes targeting integrated...ddPCR and analysis Equipment Class II, Type A2 Biological Safety Cabinet Aspirating unit Microcentrifuge...glutaGRO, Corning 25-015-CI) 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...cartridge holder with bleach and place it in the Biological Safety Cabinet (BSC). Make sure that the BSC ...

...Complete to a 15 mL conical and an appropriate amount of 25 mM chloroquine to the conical so that the final...am): Harvest virus Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette... Da Heat-inactivated FBS 1x PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...10 cm tissue culture dish, Corning 430167 15 mL conical tubes, VWR 21008-216 Hydrochloric acid Sodium hydroxide... or sodium hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment...optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI ...Start The health of the packaging cell line is critical for obtaining high viral titer. 293T cells should...

... of PBS. Calculate the fraction of fluorescent-positive cells in each well. When calculating titer, only...fluorescent cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Reagent...You Start The health of the target cell line is critical for obtaining accurate titers. Check the cells...remaining well, a cell count is required for calculating the titer. Incubate for 48–72 h. Gently aspirate...leading to underestimation of the true titer. Calculate the transduction units per mL (TU/mL) using either...

...Nuclease-free water Microcentrifuge tubes 96-well optical plate Pipette tips General Considerations Always...should also contain an internal passive reference (typically ROX dye), to normalize non-PCR–related fluorescence...Addgene. The values highlighted below in red were calculated using this plasmid, but will change if you use...use a different plasmid. Sample Calculation Size in bp of Addgene plasmid #59462 : 6208 bp Concentration...vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do NOT treat your plasmid standard with DNase..., use dilutions 5–8 Note: at Addgene, samples typically range from 1 x 10 12 GC/mL to >2 x 10 13 GC/mL...Pro-Tips The quality of the sample dilution series is critical. Make sure to pipet each dilution up and down ...

...lines of interest Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...CI) Heat-inactivated FBS 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...selective medium is used, it may have additional biological effects on the cell line. In the absence of selective... medium throughout experiments be determined empirically, and that the cell population be monitored regularly...individual cells by passing several times through a serological pipet or by passing through a 40 µm cell strainer...will be used to seed the 96-well plate. Sample Calculation First, use a hemocytometer to quantitate the ...well plate, make 10 mL of a 5 cell/mL solution. Calculate the total cells needed: Total cells needed: 10...

...for short term studies of protein function. We specifically use this protocol with a subclone of HEK293T...perform your experiment Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette... or sodium hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment...Before You Start The health of the cell line is critical for obtaining high levels of virus. HEK293T cells...optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI ...batches of PEI depending on the user, quantities of chemical used, volumes, pH adjustment etc. Consequently...Pro-Tip The ratio of µg DNA:µg PEI needs to be empirically determined. Once a batch of PEI is prepared, ...

...backbone of the two DNA fragments. This reaction, called ligation, is performed by the T4 DNA ligase enzyme...nucleotides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid...digestion . Most restriction enzymes digest DNA asymmetrically across their recognition sequence, which results...digested end of the DNA fragment. The overhangs, called "sticky ends", are what allow the vector and insert.... Use a (Link opens in a new window) ligation calculator to easily quantify how much vector and insert...of DNA, buffer and ligase into a 10μL reaction, scale the reaction size as necessary - being sure to increase...vector end. (Link opens in a new window) Ligation calculators are easily found on the web. Just enter the concentration...

...flasks). Cell stacks provide an efficient means to scale-up without having to handle a large number of T-...am): Harvest cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...Thermo Fisher, 12605010) 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...Before You Start The health of the HEK293T cells is critical for optimal AAV yield. Do not overgrow your cells...incubator for 24 h–36 h. Proceed with transfection: Calculate the amount of each plasmid needed to have a 1:... Therefore, for each plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1 μg Volume...detach easily. Transfer cells and media into 500 mL conical vessels. Rinse CS5 once with 100 mL of PBS and ...

...gene. For example, the Whitehead Institute for Biomedical Research hosts an siRNA Selection Program that... #A9414 Luria Broth Agar (LB agar) American Bioanalytical: #AB01200-02000 Ampicillin VWR: #7177-48-2. ...transfection in a 6 cm plate. The protocol can be scaled to produce different amounts of virus as needed... transfection reagent in serum-free OPTI-MEM. Calculate the amount of Fugene® and OPTI-MEM necessary given... Sigma-Aldrich: #H9268 Protamine Sulfate* MP Biomedicals: #194729 Puromycin* Sigma-Aldrich: #P8833 Target...0.5 mL for a high MOI, and 0.1 mL for a low MOI). Scale the amount of virus added depending on the size ...antibiotic Per 40 grams of powder from American Bioanalytical catalog # AB01200-02000, LB contains: 10g tryptone...

...resistant colonies Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Reagent...You Start The health of the target cell line is critical for obtaining accurate titers. Check the cells...required to kill your target cell line needs to be empirically determined. Treat the target cells with a range... can usually be omitted because this dilution typically produces a lawn of cells, which cannot be accurately...integration events decreases as the dilution increases. Calculate the transduction units per mL (TU/mL) as follows...

...analyzing the gene’s function. The process is shown graphically in the following cartoon, in which we are adding...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-...reaction and 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible...step, depending on the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...electro-competent cells instead of the more common chemically-competent cells. The number of bacterial colonies...will tell you your “background” level or more specifically it will tell you how many colonies you can expect...

...: Buffer exchange Equipment Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller...controller Benchtop centrifuge compatible with 50 mL conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2...VWR 89130-900 50 mL pipette, VWR 89130-902 50 mL conical tubes, VWR 89039-656 Microcentrifuge tubes, VWR...inhibitors to 1X if this has not already been done. We typically start with about 250 mL of supernatant and add...buffer. re Collect the flow-through in a 50 mL conical tube and dispose after use. Carefully pour the ...closure and loosen cap. Place column in a 50 mL conical collection tube. Centrifuge column at 1000 x g ... to reach the desired concentration. Pro-Tip Periodically check the concentration on the NanoDrop Spectrophotometer...

...Stack such that the electrical contacts are aligned with the corresponding electrical contacts on the blotting... 37 °C . Determine the absorbance at 590 nm . Calculate the average absorbance of the duplicate samples...vary between samples and target proteins but is typically between 10–50 µg . If the protein is in low abundance...Bottom Stack on the blotting surface. Align the electrical contacts on the blotting surface of the iBlot...concentration will vary between antibodies but is typically between 1–10 μg/mL. Incubate the membrane overnight...concentration will vary between antibodies but is typically between 1–10 μg/mL. Incubate the membrane with...

...primary antibody to use will vary and needs to be empirically determined. If possible, run an initial test ...Microplate shaker ELISA plate washer (optional) Scale Microsoft Excel or similar software Reagents Purified...Bovine serum albumin (BSA), VWR 10273-264 50 mL conical tubes, VWR 89039-656 96-well polyester (clear) ...antibody concentration will vary and needs to be empirically determined. We suggest starting with three concentrations... rpm for 15–30 min . Pro-Tip Check the plate periodically to determine when the desired color change has...curve will vary between targets and needs to be empirically determined. The dilution series created in section...don’t forget to factor in that dilution when calculating the original sample concentration!). Sample Data...

...recommend 5’ and 3’ sequencing primers. These primers typically anneal to the backbone and can help you verify...sequencing results as a trace file (.ab1) which graphically depicts the sequence as a series of colored peaks...at the trace file, you will see that these base calls are unreliable. This is an example of a trace file...apparent mismatch/mutation may be the result of a mis-called peak in the trace file. If the mutation is not...and Chromas (PC). How can I tell if a peak is mis-called? Open the trace file and use the search feature...Check the trace file and see if you can manually call the correct base at the position. Sometimes an “...

...after working in the lab. Ensure that a designated chemical waste accumulation site is present, and that you...before starting the work. Before working with chemicals, first review their material safety data sheets...waste that may be contaminated with pathogens or chemicals that present a danger to people and the environment...disposal, commonly done with an autoclave. Only mechanical pipetting should be done in the laboratory. Don... evacuate and close off the area and alert the chemical hygiene officer. BSL-2 Guidelines Remember, the...so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors...

...treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the...plasmid and 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible...step, depending on the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...electro-competent cells instead of the more common chemically-competent cells. The number of bacterial colonies...will tell you your “background” level or more specifically it will tell you how many colonies you can expect...

...Adeno-associated viral vectors (AAV). This protocol specifically uses primers and probes targeting the ITR elements... is genome copies and generally represents the physical number of viral particles. Users may need to run...biosafety regulations. Equipment Class II, Type A2 Biological Safety Cabinet, Labconco 302411100 Droplet Digital...cartridge holder with bleach and place in the Biological Safety Cabinet (BSC). Make sure that the BSC ...Pro-Tip Prepare the viral dilutions in a separate biological safety cabinet (dilution BSC) than that used ... a CSV file and use the following equation to calculate the titer for each sample: $$T = {R*C*D*1000\over...

...material, use an analytical scale. This will provide more accuracy as these types of scales display more ...are weighing by looking for a weight range on the scale. Make sure that the weight of the material you’re... balance. This will help you remove it from the scale later on as otherwise, the paper could stick to ...