...cells were 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...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 1:6 18.9 113.4 Gently add the diluted PEI...possible range of ratios to test: Ratio of DNA:PEI Amount of DNA (μg) Volume of 1 mg/mL PEI (μL) 1:1 18.9 .... Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000 Da ...stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of OptiPro SFM (per 10 cm plate). 56.7 µL of 1 mg/mL PEI...for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, replace ...prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 days ...

...determined for your sample. Typical ratios may range from 1:1 to 1:6. Add the diluted PEI-MAX to the diluted...solution at -20 °C. 1 mg/mL PEI-MAX Add 1 g of PEI-MAX powder to 900 mL deionized water in a 1 L bottle and ...to mix. Add 450 µL of 1 mg/mL PEI-MAX to the second tube of 6 mL BCD TFX (for a 1 mg/mL stock solution ...antibody At 168 hours (1 week) post-transfection, harvest the antibody. Transfer the HEK293 cells and media...February 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest...at -20 °C. Procedure Section 1: Seeding cells The day prior to transfecting, seed a 108 mL culture of HEK293.... Cap the tubes and incubate for 1 h in the 37 °C bead bath. Transfer the PEI-MAX and recombinant antibody...

...get more colonies if you use 1 μl of a 1:5 or 1:10 dilution rather than 1 μl directly.... 20-30 mins. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42...and then (optional) incubate in 37°C incubator. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 ...Introduction Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria... more easily transformed and that will help to maintain the plasmid without rearrangement of the plasmid...prepared for optimal transformation efficiencies upon thawing. For the highest transformation efficiency, we...Pro-Tips Commercial competent cells range significantly in their transformation efficiency. The lowest efficiency...

...Consortium A.2 Map of pLKO.1 A.3 Related plasmids B. Designing shRNA Oligos for pLKO.1 B.1 Determine the optimal...Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing... References H.1 Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector...marker encoded in pLKO.1 allows for convenient stable selection. Figure 1 : Map of pLKO.1 containing an shRNA...puromycin should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add appropriate...Appendix I.1. Sequence of pLKO.1 TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning...convenience. See “warranty information” in appendix. Table of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi...

...ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided high transfection efficiencies...possible range of ratios to test: Ratio of DNA:PEI μg of DNA μL of 1 mg/mL PEI 1:1 18.9 18.9 1:2 18.9 37.8... 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9 113.4 Gently add the diluted PEI mixture to the...: Monday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a...packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection. Remove media, replace...ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB...prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days ...

...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...