...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...μL Sterile dH 2 O 0.2 μL Taq DNA Polymerase (5 units/μL) PCR Machine Agarose Gel Procedure Primer Design...(PCR). Basic PCR Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double...

...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 ... of the pooled sample is below 1.0 mg/mL proceed to Option 2 with a buffer exchange/concentration using...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 ...

...worn outside of the BSL-2 area. BSL-2 laboratories must be clearly marked as “BSL-2.” The names and contact... biosafety level 1 (BSL-1) and biosafety level 2 (BSL-2). The purpose of the four levels is to distinguish...in addition to BSL-2 guidelines below, including PPE protocols . Working in a BSL-2 laboratory requires... steps to ensure you are working in BSL-1 and BSL-2 labs safely. Protocols... Biosafety Levels One and Two (BSL-1 and BSL-2) Intro to the Lab Bench Check out more protocols, videos...humans, for example, non-pathogenic E . coli . BSL-2 is for labs that work with pathogens including organisms...as Staphylococcus aureus or Vibrio cholerae . BSL-2 includes all of the precautions needed in BSL-1, along...

...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...buffer for EcoRI 1 μL EcoRI 14 μL ddH 2 O Incubate at 37°C for 2 hours. Run digested DNA on 0.8% low melting... For a standard T4 ligation, mix: 2 μL annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning ...

...150 flask (Corning, 150 cm 2 ) Five chamber cell stack (CF5, Corning, 3180 cm 2 ) Heat-inactivated FBS (... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working...after 30 passages. Procedure Trypsinize and resuspend the HEK293T packaging cells from 2 x T-150 flasks. ...from one Five Chambers Cell-Stack (CF5) (3,180 cm 2 - the same surface area as 21 x T-150 flasks). Cell...2019 Workflow Timeline Day 0: Seed cells in CF2 Day 2: Seed cells in CF5 Day 3 (am): Transfect cells Day...Aspirate PBS and add 4 mL of 0.05% Trypsin/EDTA. Wait ~2 min. Neutralize trypsin by adding 4 mL of HI-FBS. ... suspension (no clumps of cells). Pool cells from 2 x T-150 flasks. Adjust volume to 200 mL with DMEM ...

...Activation 95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold ...diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across...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 ...Detach cells by incubating with 200 µL TrypLE for 1-2 minutes. Resuspend cells in 500 µL DMEM complete and... DG8 cartridge into the cartridge holder. Using a 2-50 µL multichannel pipet, load 20 µL of the reaction...Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open the QuantaSoft software...

... 95 10 2 1 Denaturation 95 0.5 2 50 Annealing/Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold ...should decrease by a factor of 2 across the dilutions. In the example below, 2-fold serial dilutions of a ... considered biosafety level 1 but may require BSL-2 handling depending on the insert. Please ensure that...single channel pipette 1-10 µL multichannel pipette 2-50 µL multichannel pipette 20-200 µL multichannel ...20X): 5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400) Dilution... DG8 cartridge into the cartridge holder. Using a 2-50 µL multichannel pipet, load 20 µL of the reaction...Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open the QuantaSoft software...

...the solution can be stored at 4 ℃ for up to 2 months. After 2 months, discard the tube and thaw a new working...were transfected using 1:1, 1:2, 1:3 and 1:6 ug of pRosetta:ug of PEI. The 1:2 and 1:3 ratios provided high...lentiviral vectors) Day 1 (pm): Transfect Cells Day 2 (am): 18h post transfection - Remove media, replace... use. Thawed aliquots should be discarded after 1-2 months. 1 mg/mL polyethylenimine, linear MW 25,000...times a week: Monday: Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 ...culture plates. Incubate the cells at 37 ℃, 5% CO 2 for ~20h. Gently aspirate media, add 10 mL fresh DMEM...plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives...

...stock 45 uL 10x 10x Dilution 2 5uL Dil. 1 95 uL 20x 200x Dilution 3 20uL Dil. 2 80 uL 5x 1000x Dilution 4...from the production process (DNase does not penetrate the virion). 5μL sample + 39μL H 2 O + 5μL 10x DNase... AAV preparation. Workflow Timeline Plate set-up: 2 hours qPCR run: 1.5 hours Data analysis: 30 minutes...Therefore we need to dilute 1.25 μL stock into 98.74 μL H 2 0 *Pro-Tips* Once a validated standard curve is obtained...a small aliquot of each standard (enough for 1 or 2 plates) and store at -20°C. Once a standard is thawed...with transparent film. Centrifuge at 3,000 rpm for 2 min to bring the sample to the bottom of the tube....from step 3 / melt curve Example of plate set-up: 1 2 3 4 5 6 7 8 A 1.00 x 10 9 1.00 x 10 8 1.00 x 10 7 ...

...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...invert to mix. Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer to each sample. Add 2 µL 10X reducing...Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...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...

...the solution can be stored at 4 ℃ 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...packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 hours post transfection. Remove media, replace...culture plates. Incubate the cells at 37 ℃, 5% CO 2 for ~20 hours. Prepare a mixture of the 3 transfection...plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives...DNA:PEI μg of DNA μL of 1 mg/mL PEI 1:1 18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5...polyethyenimine (PEI) transfection protocol. This procedure can be modified for alternative packaging cell...

...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 ℃. 1x PBS-MK buffer Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume ...need more time, you can alternatively centrifuge for 2 h at 200,000 g at 18 ℃. Carefully take the QuickSeal...interface of the 60% and 40% gradient (see Figure 2) with an 18 g needle. Place the first microcentrifuge...at first and will progressively slow down. Option #2 Puncture the QuickSeal tube at the bottom using an...

...transfer, block, incubate with primary antibody Day 2: Incubate with secondary antibody Video Watch this... Microcentrifuge 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...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...

... glacial acetic acid 57 mL of dH 2 O Store Solution III at 4°C Grow 2 mL overnight cultures from single...Resuspension buffer Denaturing solution Renaturing solution 2 mg/mL RNase A TE or water-saturated phenol-chloroform...100 μL of cold Solution I. Vortex the solution for 2 min or until all bacteria are fully resuspended. Add...pipetting or carefully pouring. (Optional) Add 5 μL of 2 mg/mL RNase A to the supernatant in the new tube and... to the recovered aqueous DNA layer. Repeat steps 2-4. Note: Phenol-chloroform is a hazardous waste - ...: Ethanol Precipitation To your DNA solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of...plasmid DNA. This page will discuss the general procedure for purifying plasmid DNA from bacterial culture...

...Culture should be between 1.5–2 x 10 6 cells/mL with >95% viability to proceed with transfection. Transfect...Warnings HEK293 cells are considered biosafety level 2. Please ensure that you are in compliance with your... 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest...conical tubes Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 ...250 mg Benzamidine 25 mL Aprotinin saline solution (2 mg/mL) Mix well and sterilize through a 0.2 µm PES... a 500 mL vented flask.Incubate in a 37 °C, 5% CO 2 incubator on a shaking platform set to 120 rpm. *Pro-Tip... not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability...

...method 1) or the volume of virus (method 2): Method 1 Method 2 $$T = {N*F*D\over V_T}$$ Where: T = Titer...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your...of lentiviral vectors) Day 1: Transduce Cells Day 2 (am): Remove media, replace with fresh media Day 4...complete. Mix well by pipetting or inverting. Aliquot 2 mL of cell suspension into each well of the 6-well... supernatants to multiple freeze-thaw cycles. Procedure Seed 75,000 cells into each well of a 6-well dish...

...each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 ...cells you are using). Put the tubes back on ice for 2 min. Add 250-1,000 μl LB or SOC media (without antibiotic...instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before heat-shock Shorten or skip the ...DNA Ligation Introduction Transformation is the process by which foreign DNA is introduced into a cell.... competent cells. This is a relatively simple procedure and is useful for performing low efficiency transformations...media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw on ...competent cells) to ensure that your transformation procedure is working. TIP: Sometimes less is more. Although...

...temperature between 50-60°C for your PCR primers. Step 2: Linearize Vector In this example, the vector is cut...20-30 Eluted DNA 10-50 ng/μl 1 dGTP (100mM) 2.5 mM 2 DTT (100 mM) 5 mM 1 BSA (10 μg/μl) 0.25 μg/μl 1 T4...your treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing... reaction is now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, ...together through the transformation/replication process. LIC employs long overhangs to form a stable association...free nucleotides from your PCR product before proceeding, as they will interfere with the exonuclease ...

...agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel). Mix agarose powder with...gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds...allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases...Introduction Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base...Ethidum bromide (stock concentration of 10 mg/mL) Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g... from Your Gel: If you are conducting certain procedures, such as molecular cloning, you will need to ...

...RNAzol®, QIAzol® (for Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl....5% (wt/vol) N-laurosylsarcosine (Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as...recipe). If using TRIzol®, jump down to the Option #2 - TRIzol® Protocol section below. Homogenize or lyse...following sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion...aliquots of it and storing those in -80°C. Option #2 - TRIzol® Protocol Homogenize or lyse tissues or cells...by hand for 10 seconds. Incubate the sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at ...with the volatile reagents in the list above. Procedure Option #1 - Solution D Protocol Before starting...