...Dilution 1:10 100 of Stock Virus 900 150 1,350 1:100 1:100 100 of 1:10 900 150 1,350 1:1,000 1:1,000 100...100 of 1:100 900 150 1,350 1:10,000 1:10,000 100 of 1:1,000 900 150 1,350 1:100,000 1:100,000 100 of 1... 1:10,000 900 150 1,350 1:1,000,000 Mix the dilutions well Note: the 1:10 dilution can usually be omitted...with 1 mL of 0.1% crystal violet for 10 min at RT. Gently remove the stain. Wash cells 3x with 1 mL of...multiple dilutions. Sample Data Figure 1: A549 cells were transduced with the indicated serial dilutions ... media from the wells. Gently wash the cells with 1 mL of PBS and aspirate wash. Filter 0.1% crystal violet...volume in the well (mL) x dilution factor e.g., If the 1:100,000 well has 75 colonies, then there are 75 colonies...

...tissues: use 1 mL of Solution D per 100 mg of cells. For cultured cells: use 1 mL of Solution D per 1 X 10 7...tissues: use 1 mL of TRIzol® per 100 mg of cells. For cultured cells: use 1 mL of TRIzol® per 1 X 10 7 cells...Option A): Add 1 volume of Isopropanol to the extracted aqueous layer. Incubate at -20°C for 1 hour. Lithium.... Add 0.2 mL of Chloroform/Isoamyl alcohol (49:1) per 1 mL of TRIzol® used. Shake vigorously by hand for... sample(s).Transfer tissue/cell lysate to a 4 mL tube. Add the following sequentially to 1 mL of lysate... User Guide from ThermoFisher Scientific . Figure 1: A diagram of the different steps in RNA extraction...

...Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400)...95 10 2 1 Denaturation 95 0.5 2 50 Annealing/Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold 4 ...Use a single channel 1–10 µL pipette to add 5 µL of each viral sample to Dilution 1 in the 48-well dilution... 95 µL 1X PCR buffer (1:8,000) Dilution 4 (20X): 5 µL in 95 µL 1X PCR buffer (1:160,000) Dilution 5 (20X...95 µL 1X PCR buffer (1:3,200,000) Dilution 6 (2X): 50 µL in 50 µL 1X PCR buffer (1:6,400,000) Dilution ...50 µL 1X PCR buffer (1:12,800,000) Dilution 8 (2X): 50 µL in 50 µL 1X PCR buffer (1:25,600,000) Use multichannel...pipettes for the dilution series. For dilutions 1–5, use the 1–10 µL multichannel pipette set to 5 µL. For...

... Proceed with transfection: Calculate the amount of each plasmid needed to have a 1:1:1 molar ratio with...7.5% Sodium Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose. 1 mg/mL polyethylenimine (PEI) ... determine the total μg/bp we need to achieve a 1:1:1 molar ratio of each plasmid: 2000 μg / 24,961 bp...deionized water + 100 mL of 1 M Tris HCl pH 8.5 + 60 mL of 5 M Sodium Chloride + 4 mL of 1 M Magnesium Chloride... adherent cells) 1 mg/mL Polyethylenimine (PEI) 25 kDa MW Pro-Tip Other transfection reagents may be used...high glucose, Corning 10-013-CV DMEM, low glucose (1 g/L) glucose, sodium pyruvate, Corning 10-014-CV (... sodium bicarbonate, Corning 25-035-CI (optional) 1 M HEPES, HyClone SH30237.01 (optional) L-alanyl-L-glutamine...

...NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH to 7.0 Autoclave or filter sterilize 1 M sodium phosphate...NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH to 7.0 Autoclave or filter sterilize 1 M of sodium phosphate...protease inhibitor cocktail. Add 1 part Protein A/G binding buffer to 1 part tissue culture supernatant...Choose Option 1 or Option 2 based on the concentration of the pooled sample above. Option 1: Buffer exchange...antibody to 1 mg/mL with PBS if needed. For long term storage, add sterile sodium azide to 1 mM. Option...purify recombinant antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment...°C bead bath Clamp stand and clamps Autoclave 0.1–1 mL single channel pipette 0.5–10 µL single channel...

...polybrene (μL) 1:10 150 1348.5 1.5 1:25 60 1438.5 1.5 1:50 30 1468.5 1.5 1:75 20 1478.5 1.5 1:100 15 1483.5...Factor V T = Transduction Volume, mL For example: If 150,000 cells were transduced in the 1:100 well, resulting...dilutions. Sample Data Figure 1: 293T cells were transduced with a range of dilutions of pHAGE-TO-dCas9...Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with...Calculate the transduction units per mL (TU/mL) using either the dilution factors (method 1) or the volume...Method 1 Method 2 $$T = {N*F*D\over V_T}$$ Where: T = Titer, TU/mL N = Number of cells transduced F = Fraction...for 48–72 h. Gently aspirate media and replace with 1 mL of PBS. Calculate the fraction of fluorescent-positive...

...dilution Dilution 1 (DNase step) 5 uL AAV stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X Dilution...reference virus that is 1 x 10 13 GC/mL. We recommend always using a reference within 1-log of the expected...DNase buffer + 1 μL DNase Gently mix sample (do not vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical...dilutions 5–8 Note: at Addgene, samples typically range from 1 x 10 12 GC/mL to >2 x 10 13 GC/mL and we use...Equipment qPCR instrument Heating plate Pipettors 1–10 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette...The reference material should have a titer within 1-log of the expected titer of the samples being tested...

...95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold 4 ... primers/probe (FAM) 1 µL 9 µL 900 nM, 250 nM 20X RPP30 primers/probe (HEX/VIC) 1 µL 9 µL 900 nM, 250 ...Update: July 7, 2023 Workflow Timeline Day 1: Seed and transduce cells Day 4: Treat cells with Benzonase ...a well of a 6-well plate (1 dilution per well). Leave one well untransduced (add 150 µL of DMEM complete...with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete and transfer to a microcentrifuge...4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open the QuantaSoft software ...14440 24260 1.190436933 5.95E+06 7.29E+06 7 Untransduced 1 6.4 17940 0.0007134894091 1.43E+02 Table: Example...

...mL polybrene (µL) 0 0 500 1:5 300 200 1:10 150 350 1:50 30 470 1:100 15 485 1:500 3 497 Add 0.5 mL of a... if an MOI >1 was used, some cells may have 1 copy of the transgene, while others have >1 copy of the ... Cas9. A549 cells were transduced with lentiCas9-Blast and then selected with 1 µg/mL blasticidin for... well by pipetting or inverting the tube. Aliquot 1 mL of cell suspension (i.e., 50,000 cells) into each... early polyclonal populations. Sample Data Figure 1: Generation of monoclonal cell lines from expansion...Different brands and FBS lots can promote or inhibit transfection. Test a variety of brands and FBS lot...-term protein expression observed using transient transfection approaches, generating cell lines using...

...June 14, 2023 Workflow Timeline Day 1: Run SDS-PAGE and stain gel Day 1 or later: Image analysis Video Watch...lane Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 ...antibodies using Coomassie stain. Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel...deionized water. Gently invert to mix. Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer to each sample...attach to a power supply. Run the gel at 150 V for 1 h . Pro-Tip If the samples are running unevenly and...Add 20 mL of SimplyBlue SafeStain and incubate for 1 h with gentle agitation on a rocking platform. Pour...sink. Add 100 mL of deionized water and incubate for 1 h with gentle agitation on a rocking platform. Pour...

...cells transduced with lentiCas9-Blast 1 . A549 cells were transduced (MOI = 37) and selected with 1 μg/mL...Cas9. A549 cells were transduced with lentiCas9-Blast 1 and then selected with 1 μg/mL blasticidin for...Because this is such a small volume, first make 1 mL of a 1:100 dilution of the homogenized cell solution... solution, transfer 12.5 µL of the 1:100 dilution to 10 mL of conditioned medium. Transfer 100 µL of the... the highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal...conditioned medium (see below for more details) Day 1: Seed individual cells in a 96-well plate Day 2–14...cells will appear as colonies in the well ( Figure 1 ). You will be able to tell if there was more than...

...Vented Falcon Tubes at 30-37 ℃, 225 rpm for 1 hour. After the 1 hour shaking period, pool and gently mix ...Timeline Day 1: Transform, recover, set up overnight growth (Estimated time 2-3 hours) Transformation should... (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives include Stbl4 cells or other ultra-high...0.1 cm ) 20 mL SOC recovery media (Lucigen, 80026-1) 8X LB Agar + Antibiotic 245 mm bioassay plates (Molecular...into each of four 14 mL Vented Falcon Tubes and have 1 mL SOC per electroporation readily available for post-electroporation...autoclaved, sterile reagents for all steps. Procedure Day 1 Add 200 ng DNA to each 50 µL aliquot of thawed Endura...: Bio-Rad Micropulser Ec1 0.1 cm cuvette, 1.8 kV, 1 pulse. Aliquot 25 µL DNA-Endura into pre-chilled cuvette...

...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 DNA polymerase...6: Anneal and Transform Mix your treated vector and insert at a molar ratio of 1:2 or 1:3, using between...now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, following a standard...collection of empty LIC cloning vectors Protocol Step 1: Design Your Primers Primer design for LIC is often...reaction for 5 minutes at room temperature, then add 1 μl of 25 mM EDTA, followed by another 5 minutes at...stability to hold the plasmid together through the transformation/replication process. LIC employs long overhangs...association between fragments, allowing for transformation without ligation. Because of its dual polymerase...

...avoid breathing in the vapors. Workflow Timeline Day 1: Antigen Coating Day 2: Blocking Day 3: Primary antibody... Spectrophotometer compatible with 96-well plates 1–10 µL single channel pipette 2–20 µL single channel... µL, VWR 76322-134 Pipettes, 10 mL, VWR 89130-898 1 L polystyrene bottle, Corning 430518 PBS, 1X pH 7.4...Warm reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard and coat the plate Dilute...stock into 900 µL PBS in a microfuge tube and vortex. 1 ng/µL : Add 450 µL of 2 ng/µL stock into 450 uL PBS...microfuge tube and vortex. 0.5 ng/µL : Add 450 µL of 1 ng/µL stock into 450 µL PBS in a microfuge tube and... a microplate shaker set at 400 rpm and shake for 1 min . Repeat steps 8–10 twice for a total of three...

...chemiluminescence substrate by mixing 1:1 reagent A to reagent B. Gently incubate the membrane in the chemiluminescence...Block the membrane in blocking buffer for 1 h at RT on a shaking platform. Wash the membrane 3x for 5 ...24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer, block, incubate with primary...supernatant. Resuspend cell pellet in 1 mL of 1X PBS and transfer to a microcentrifuge tube. Centrifuge...antibodies but is typically between 1–10 μg/mL. Incubate the membrane overnight in primary antibody at 4...antibodies but is typically between 1–10 μg/mL. Incubate the membrane with secondary antibody for 60 min...primary antibodies raised in a mouse. Procedure Section 1: Lyse cells Centrifuge 5 x 10 6 cells for 5 min at...

... resistance) Procedure Day 1 Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium... than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve of Transformed E. coli after...lawn of E. coli and no apparent selection. 150 µL of 1 mg/mL Carbenicillin plated over-agar Plate shows several...over-agar Plate shows less individual colonies than the 1 mg/mL plate and effective selection. 150 µL of 4 mg...incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest. See our transformation page...for a detailed heatshock transformation protocol. Plate 50 µL of transformed E. coli /rescue media suspension...also like... Making LB Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture...

...(PPE) for BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL-1 and BSL-2 labs...Levels One and Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the... T4 polymerase pLKO.1 - TRC Cloning Vector Cloning protocols for using the pLKO.1 vector, a backbone used...practical lab protocols that you can use for a wide range of applications, with videos for select protocols...Ligation Assemble DNA using DNA ligase Bacterial Transformation Introduce DNA into a bacterial strain Watch...Virus Name Description Link to Video General Transfection Introduce plasmid DNA to mammalian cells Lentivirus...Produce lentivirus with a polyethyenimine (PEI) transfection protocol Fluorescence Titering Assay for Lentivirus...

...restriction digestion reaction could look like this: 1 µg DNA 1 µL of each Restriction Enzyme 3 µL 10x Buffer...definition: one unit of enzyme will cut 1 µg of DNA in a 50 µL reaction in 1 hour. Using this ratio, you can ..., incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient...ng of DNA, while molecular cloning often requires 1 µg of DNA. The total reaction volume usually varies...tube at appropriate temperature (usually 37 °C) for 1 hour. Always follow the manufacturer’s instructions...sufficient. For digests with >1 µg of DNA used for cloning, it is recommended that you digest for at least...

...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can...now bind to the primer DNA sequence. Extend DNA for 1 minute at 72°C: The Taq polymerase has an optimal ...DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 mM each nt) 2.5 μL Forward Primer ...primer melting temperature (Tm). Set extension step at 1-2 minutes per kilobase of product depending on whether...working concentration of each primer (10uM) by making a 1:10 dilution of the stock. For example, add 100µl of...step heats the double stranded DNA template strand to the point where the strands start denaturing and ...

...Preparation 1 M NaCl/PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS ...gradient for AAV purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note...Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing... step: mix 4.5 mL of 60% iodixanol and 13.5 mL of 1 M NaCl/PBS-MK buffer 25% iodixanol step: mix 5 mL ...Hamilton needle, taking care to avoid bubbles (Figure 1). 8 mL of 15% iodixanol step 5 mL of 25% iodixanol...disturb the gradient!** Collect Fractions Option #1 Prepare a row of roughly 20 open 1.5 mL microcentrifuge...with a 16 ga needle and start collecting 0.5 mL to 1 mL fractions per tube. Avoid the proteinaceous material...

...antibody concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL of the diluted antibody...antibody concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled secondary...Last Update: January 20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment...into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-D-lysine coated...with a laboratory wipe to remove excess liquid. Add 1 drop of anti-fade mounting medium to the microscope... Inclusion of this information is solely for transparency intended to support reproducibility in science...you know expresses the protein, such as cells transfected with a plasmid to express the protein of interest...

...situations where the 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in...in ligation or transformation reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert to vector...the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will work just fine. We recommend...Restriction Digest of Plasmid DNA Bacterial Transformation Background Information The final step in the...the backbone and the complete plasmid can be transformed into bacterial cells for propagation. The majority...recognition sequence, which results in a single stranded overhang on the digested end of the DNA fragment... incubation at 37°C. Proceed with bacterial transformation . Tips and FAQ Do controls When doing ligations...

...10 mg/mL) Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose. Pro-Tip Agarose gels are...different buffers and do not use water). Microwave for 1-3 min until the agarose is completely dissolved (but...samples. Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading...the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and ...the solution has a tendancy to boil over. Placing saran wrap over the top of the flask can help with this...are usually referred to as ‘bands’ due to their appearance on the gel. Pro-Tip If you will be purifying ...

...from casein 10.0 g sodium chloride 12.0 g agar-agar 1 L Sterile H 2 O Sterile plates of your desired size...bottle for autoclaving. We make 400 mL of agar in 1 L bottles and 200 mL of agar in 500 mL bottles. The...measure out 100 mg of ampicillin powder, add it to 1 mL of water, dissolve by vortexing, and filter sterilize... You should not store your plates for longer than 1 month at any temperature and should always check for... for contamination prior to use. Negative Result 1: Both Strains Grow Assuming the appropriate strains...x (0.220 L) = 8.14 g pre-mixed LB-agar powder. Transfer the LB-agar powder you’ve measured out into an...molten agar from boiling over in the autoclave. Transfer the sterile water (in our case 220 mL) to the ...

...fragment that needs to be amplified should be within 1-10 kB in size. The structure of the primer should ...18-24 bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer...complementary to template strand). However, primers do not need to correspond to the template strand completely; it...primer corresponds completely to the template DNA strand so elongation can proceed. Usually a guanine or...

... by PCR has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent ...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...recipient plasmid backbone. Transformation Proceed with the transformation according to the manufacturer...from your transformation will give you the first indication as to whether your transformation worked. Your...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary PCR based cloning is incredibly versatile...chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may need to increase...

... as shown in the diagram above, to create streak #1. Pro-Tips Hold your tooth pick at an angle, the way... or freshly sterilized loop, drag through streak #1 and spread the bacteria over a second section of the...also like... Making LB Agar Plates Bacterial Transformation Recovering Plasmid DNA from Bacterial Culture...

...annealing can be achieved by one of two methods: Method #1 Place the mixed oligos in a 1.5mL microfuge tube. ...vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard ligation reaction (ex. to ligate an...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary Oligo overlap cloning can be used anytime...the vector with 0.75-6 ng of annealed oligos). Transform 2-3μL into your favorite competent bacteria and...

...dispense small amounts of liquid (think: 0.1 µL to 1 mL). When working in a laboratory, properly dispensing...right. Pipette Dispense Volume P2 0.2 to 2 µL P10 1 to 10 µL P20 2 to 20 µL P100 10 to 100 µL P200 20 ...and the third (red) digit is hundredths. Therefore, 1 µL would read as 100 (as shown in the picture above...boxes that the tips come in often indicate a volume range that the tip can hold. This should give you an idea...

...Figure 1 depicts an image of a compound light microscope with the main components labeled: Figure 1: Diagram...your cell culture dish. Microscopes come in a huge range of shapes and sizes - from phone-sized, foldable...powerful (but are cheap and accessible) to massive transmission electron microscopes that allow us to see cellular...

...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent ...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...self-ligating recipient plasmid backbone. Transformation Transform your ligation reaction into your bacterial...from your transformation will give you the first indication as to whether your transformation worked. our...Digest of Plasmid DNA DNA Ligation Bacterial Transformation Summary The following technique can be used...conduct a positive control to ensure that your transformation worked. You should also verify that you are...

...If using a NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal. Close the...spectrophotometer. A spectrophotometer uses the absorbance/transmission of light through a liquid to determine the concentration...measured under the 260/280 column (A good purity ranges from 1.80-2.00). Repeat for each sample. Notes:...

...resuspended in a small volume (e.g. 0.02 g to resuspend in 1 mL), you may place a microcentrifuge tube on the balance...material that you are weighing by looking for a weight range on the scale. Make sure that the weight of the material... material you’re weighing out is within this range. If you need less than a gram of material, use an analytical...cross-contamination with other substances. They also help you transfer the material to your tube or container. Pro-Tip...weighed out the correct amount of your reagent, transfer it into your container. Make sure that any spills...

...ssDNA. (Rabe & Cepko, 2020). Incubate the mix for 1 hour at 50 °C or follow manufacturer's instructions...of a Gibson Assembly is a fully ligated double-stranded DNA molecule. This has proven to be an efficient...for the reaction: T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5...fragment. Pro-Tip Add Extreme Thermostable Single-Stranded DNA-Binding protein (ET SSB) to the isothermal...You can purchase master mix or make your own. Transform bacteria with the DNA and screen for the correct...

...insert, but significantly off center (ideally around 1/3 of the way from one end), and also cuts in the backbone... bacterial endonucleases that recognize a large range of DNA sequences. Given the variety of these enzymes...

...solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8). Invert the microfuge...Bacterial Culture You may also like... Bacterial Transformation Agarose Gel Electrophoresis Agarose Gel DNA...much as several miligrams and at concentrations ranging from 150 ng/μL to several μg/μL. The protocol below...optional) Reagents Overnight culture of bacteria transformed with your plasmid Resuspension buffer Denaturing... a pancreatic ribonuclease that digests single-stranded RNA. Optional: Perform phenol-chloroform extraction...