...3319, 3180 cm 2 Cellstack 2, Corning 3269, 1272 cm 2 Heat-inactivated FBS (HI-FBS) *Pro-Tip* Different... 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl 2 Add the following to the 2 L sterile... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... mL of PBS. Aspirate PBS and add 2 mL of 0.05% Trypsin/EDTA. Wait ~2 min. Neutralize trypsin by adding...Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day 7 (am): Harvest cells Equipment...times to obtain a single cell suspension (no clumps of cells). Pool cells from 2 x T-175 flasks. Adjust ...touching the cells when replacing media. T-175 flask, Corning 430825, 175 cm 2 Cellstack 5, Corning 3319...

...Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day 14–30: Analyze...colony size. Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with ... CO 2 incubator Pipet controller Hazardous waste container Microscope Hemocytometer or other cell counter... is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should be seeded in 10 mL DMEM complete, which...transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines from expansion of...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...of a 5 cell/mL solution. Calculate the total cells needed: Total cells needed: 10 mL × 5 cells/mL = 50...

...surviving cells. Even in the absence of cell death, the cell media should be changed every 2–3 days to... Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, replace with fresh media...into two 75 cm 2 flasks, etc. *Pro-Tip* This selection method results in a polyclonal cell population, meaning...Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container... the cell media. Cell death by some cells in the culture may adversely affect the surviving cells in the...media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute...

...a. Plate target cells and incubate at 37°C, 5% CO 2 overnight. Day 2: b. Target cells should be approximately...plates and grow at 37°C, 5% CO 2 overnight. Day 2: b. The target cells should be approximately 80-90% ...materials D.2 Screening for inserts E. Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol...Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty...VWR: #7177-48-2. Use at 100 μg/mL. Carbenicillin VWR: #80030-956. Use at 100 μg/mL. C.2 Annealing Oligos...oligo 5 μL Reverse oligo 5 μL 10x NEB buffer 2 35 μL ddH 2 O Incubate for 4 minutes at 95°C in a PCR machine...buffer 1 1 μL AgeI add ddH 2 O to bring to 50 μL final volume Incubate at 37°C for 2 hours. Purify with Qiaquick...

... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high...Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask... transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 days after ...tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Gently aspirate media, add 10 mL... 2 (am): 18 h post transfection - Remove media, replace with fresh media Day 3 or more (am): Observe fluorescence...Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette...

...Activation 95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold ...copies of RPP30 in diploid cells, thus the reason for multiplying by 2. $$V = 2*{copies\ RRE \over copies...diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across...each well. Detach cells by incubating with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your...channel pipette 200–1000 µL single channel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ... DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel pipet, load 20 µL of the reaction...

... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided... 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection... transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after ... applications such as stable-cell line generation. Last Update: August 2, 2023 Workflow Timeline Day 0...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...

...phosphate monobasic monohydrate (NaH 2 PO 4 ∙H 2 O), pH 7.0 138 g NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust...sodium phosphate dibasic (NaH 2 PO 4 ), pH 7.0 142 g of NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH...monobasic monohydrate (NaH 2 PO 4 ∙H 2 O) 610 mL of sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to...recombinant antibody. Section 2: Buffer exchange Choose Option 1 or Option 2 based on the concentration ...antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment Class II, Type...conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 ... channel pipette 20–200 µL single channel pipette 2–20 µL single channel pipette Reagents Aspirating pipette...

...method 2): Method 1 Method 2 $$T = {N*F*D\over V_T}$$ Where: T = Titer, TU/mL N = Number of cells transduced...Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with ...,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2 mL of cell suspension...fluorescent cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 ...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...added to 150,000 cells resulted in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent...

...protein from cells or tissue samples. Equipment Microcentrifuge 0.5–10 µL single channel pipette 2–20 µL single...transfer, block, incubate with primary antibody Day 2: Incubate with secondary antibody Video Watch this...Heat block Mini gel tank chamber Power supply iBlot 2 Gel Transfer Device Roller Spatula Platform shaker...running buffer Prestained protein ladder Ethanol iBlot 2 PVDF Mini Stack, Thermo Fisher IB24002 20X TBS Tween...immediately or store at -80 °C until ready to use. Section 2: Determine the total protein concentration and prepare...in deionized water. To prepare 20% ethanol, dilute 2 mL of ethanol into 8 mL of deionized water and mix...the transfer sandwich as follows: Unseal the iBlot 2 PVDF Mini transfer stack. Set the Top Stack to one...

...Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest antibody Equipment...Pro-Tip* Do not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability... (EBNA). Safety Warnings HEK293 cells are considered biosafety level 2. Please ensure that you are in ...compatible with 50 mL conical tubes Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to...density of 0.9 x 10 6 cells/mL in a 500 mL vented flask.Incubate in a 37 °C, 5% CO 2 incubator on a shaking ...Pro-Tip* Culture should be between 1.5–2 x 10 6 cells/mL with >95% viability to proceed with transfection...250 mg Benzamidine 25 mL Aprotinin saline solution (2 mg/mL) Mix well and sterilize through a 0.2 µm PES...

... Workflow Timeline Day 0: Seed and Transduce Cells Day 2: Replace media with fresh media containing selection...for ~2 weeks. All of the cells in the untransduced (negative) control well should be killed and no colonies...with 0.1% crystal violet and counted. Figure 2: A549 cells were transduced with the indicated serial dilutions...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...disturb the colonies. Count the colonies for at least 2 of the dilutions. *Pro-Tip* The higher dilution wells...1,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute 7,000 cells into...

...follows: Figure 2 Using the box tool, draw a box around the entire first gel lane (as in Figure 2). Select Analyze... Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 (HC)...choose Use Equation . Select the Show R 2 checkbox. *Pro-Tip* The R 2 of the trendline should be between 0.95...Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... Add 5 µL of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample...bottom part of the gel where dye is visible. Section 2: Staining the Gel Place the gel in a plastic tray ...imaging system. Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa corresponding to...

...RNAzol®, QIAzol® (for Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl...to the Option #2 - TRIzol® Protocol section below. Homogenize or lyse tissues or cells in Solution D. ...those in -80°C. Option #2 - TRIzol® Protocol Homogenize or lyse tissues or cells in TRIzol® or a similar....5% (wt/vol) N-laurosylsarcosine (Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as...following sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion...by hand for 10 seconds. Incubate the sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at ... D per 100 mg of cells. For cultured cells: use 1 mL of Solution D per 1 X 10 7 cells. Allow sample(s)...

...BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL-1 and BSL-2 labs Watch the...Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the Video! Water...Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence into a cell line Isolating...Isolating a Monoclonal Cell Population by Limiting Dilution Generate monoclonal cell lines from a polyclonal...polyclonal pool of stable cells AAV Production in HEK293 Cells Produce adeno-associated virus to deliver your...Extraction Without a Kit Extract RNA from tissues or cells without a commercial kit Plasmid Cloning Name Description...Transfection Introduce plasmid DNA to mammalian cells Lentivirus Production Produce lentivirus with a ...

...purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note : Both steps... 7.4 1X PBS-MK buffer 100X Pluronic-F68 NaCl MgCl 2 KCl Centrifugal filter units (MWCO 100 kDa) Reagent...PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS in a final volume of...at 4 °C. 1X PBS-MK buffer Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume ...(C) (formulation buffer) Add 5 mL of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation ...need more time, you can alternatively centrifuge for 2 h at 200,000 x g at 18 °C. Carefully take the QuickSeal...interface of the 60% and 40% gradient (see Figure 2) with an 18 ga needle. Place the first microcentrifuge...

...PCR tube Ice Bucket 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ... reagents on ice): 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ...normally sterile dH 2 O. To make a 100uM stock of any primer, add a number of µl of dH 2 O equal to the number...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can ...work adequately. Divalent cations such as Mg 2+ and Mn 2+ stabilize the buffer solution. These cations...(PCR). Basic PCR Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double...target DNA strand accurately and rapidly. Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72...

...each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 ...depending on the competent cells you are using). Put the tubes back on ice for 2 min. Add 250-1,000 μl LB...the competent cells in your hand instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before...commonly referred to as 'competent cells.' Many companies sell competent cells, which come frozen and are prepared...that came with your competent cells. *Pro-Tip* Commercial competent cells range significantly in their ...SOC media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw...use electro-competent cells. Instead of relying on the heat-shock to cause the cells to take up the DNA,...

...HeLa cells per well. Allow the HeLa cells to grow to the desired density before labeling. Section 2: Fixing...coated coverslips HeLa cells 24-well plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI...Fluorescent microscope 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...of the diluted antibody to the wells and incubate 2 h at RT . Remove the primary antibody and dispose ...detect antigens in cells. Here we describe the basic steps for fixing and labeling cells in culture with ...2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment Pipette controller Pipette... Seeding cells Place a sterile poly-D-lysine coated coverslip in each well of a 24-well cell culture treated...

...mL glass bottle: 4 g NaCl 4 g Tryptone 2 g Yeast Extract and dH 2 O to 400 mL Note: If your lab has pre-mixed... to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might...might want to use as much as a liter of LB in a 2 L Erlenmeyer flask. Using a sterile pipette tip or toothpick...bacterial cell. Large plasmids usually have a low copy number (approximately one or two copies per cell) and... can be present in large numbers, 50 or more per cell and have a high copy number. High copy number plasmids...increase this to 350 - 400 rpm to obtain a higher cell density. Reference Page | Top | Index...