...cloning, and titering and testing your viral preparations. ...Protocols for titering and testing your virus preparations. Antibodies Protocols for common antibody applications... Video! Inoculating a Liquid Bacterial Culture Prepare and grow bacteria in liquid medium Watch the Video...long term use Watch the Video! DNA Purification Miniprep, phenol-chloroform extract, and precipitate DNA...Ultracentrifugation Purify adeno-associated virus from a preparation produced in mammalian cells Watch the Video! ...physical titer of your adeno-associated virus preparation Watch the Video! AAV Titration by ddPCR Use ddPCR...physical titer of your adeno-associated virus preparation Lentivirus Titration by ddPCR Use ddPCR to measure...

...optimized. Plasmids for transfection: pHelper; pRC (Rep-Cap), plasmid expressing your gene of interest Triton...handled carefully. Avoid touching the cells when replacing media. T-175 flask, Corning 430825, 175 cm 2 Cellstack...mM Tris HCl, 150 mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine...et al., 2018 (Link opens in a new window) ). To prepare D1 + 0.1 M sorbitol, add 57 mL of 1.46 M sorbitol...bp) DNA Concentration (μg/μL) Volume of DNA (μL) RepCap 7,265 1.00 727.6 pHelper 11,854 1.00 1,185 Transfer..., for each plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1 μg Volume Needed... toward the cap. If the media touches the cap, replace it with a new one before putting the CS5 in the...

... ensure representation is maintained. Maxipreps - Less is more: Do not overload the Maxipreps as yield...Conical tubes (Fisher, 14-432-22) Reagent Preparation Prepare, sterilize, and pour all LB Agar + Antibiotic...individual Maxipreps by Nanodrop, Picogreen, or Qubit. Each different DNA measurement type can report slightly...quantities of library for experimental applications. Repeated amplifications should be avoided as best as possible...and plasmid recombination can all impact the representation of individual plasmids in the pooled library...required for bacterial propagation (origin of replication and antibiotic selection). This recombination... high efficiency (1x10 10 cfu/µg) commercially prepared electrocompetent cells is strongly recommended...

...Self-inactivating long terminal repeat. f1 ori f1 bacterial origin of replication. Amp R Ampicillin resistance...pUC ori pUC bacterial origin of replication. 5’LTR 5’ long terminal repeat. RRE Rev response element. A.3...Avoid stretches of 4 or more nucleotide repeats, especially repeated Ts because polyT is a termination signal...AgeI. Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA) 5 μL 10x NEB buffer 1 1 μL AgeI add...use a miniprep kit to obtain DNA. 3. Conduct a restriction digest with EcoRI and NcoI: 1 μg miniprep DNA...transfection reagent. Replace with 5 mL fresh DMEM + 10% FBS + penicillin/streptomycin. Pipette the media...of this vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC...

...polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously filtered) Microcentrifuge...conical tubes, VWR 21008-216 Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM...This protocol can be used to titer lentiviral preparations that confer antibiotic resistance. Protocols...This protocol can be used to titer lentivirus preparations that confer antibiotic resistance. Determining...Determining the titer of your lentiviral preparation allows you to control the multiplicity of infection (MOI...Timeline Day 0: Seed and transduce cells Day 2: Replace media with fresh media containing selection reagent... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...

...cells, increases reproducibility, as it eliminates the variation associated with repeated transient transfection...polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously filtered) Microcentrifuge...long-term protein expression studies. Moreover, repeating experiments in a stable cell line, as opposed ...and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing selection reagent Day...15 mL conical tubes, VWR 21008-216 Lentivirus preparation Appropriate antibiotic for selection (e.g. puromycin...puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (... after 20-30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...

...the prep with full (genome containing) particles. On average, iodixanol-purified AAV preparations contain...ultracentrifugation to get pure adeno-associated virus (AAV) preps for use in your research. Protocols...and unlike other density gradient media, it is reportedly not necessary to remove iodixanol prior to use...separate out contaminants from an impure AAV preparation. The 15% iodixanol step has 1M NaCl to destabilize...iodixanol solution is available under the name OptiPrep (Link opens in a new window) from Sigma-Aldrich...Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep (60% Iodixanol) QuickSeal tubes QuickSeal tube spacers...Centrifugal filter units (MWCO 100 kDa) Reagent Preparation 1 M NaCl/PBS-MK buffer Dissolve 5.84 g of NaCl...

...determination. Prepare a 50:1 Reagent A to Reagent B dilution of the BCA assay. Prepare serial dilutions...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. General Guidelines Acrylamide percentage...Update: January 24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer, block, incubate... Determine the total protein concentration and prepare the lysate for SDS-PAGE Determine the protein concentration...need to load a greater amount of total protein. Prepare sample for loading as follows: Add 10% v/v β-mercaptoethanol...samples for 10 min at 100 °C . Section 3: SDS-PAGE Prepare the precast gel as follows: Remove the gel from...

...polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously filtered) Microcentrifuge...conical tubes, VWR 21008-216 Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM...Protocol that can be used to titer lentiviral preparations carrying a fluorescence marker. Protocols...This protocol can be used to titer lentiviral preparations that carry a fluorescence marker. Determining...Day 1: Transduce cells Day 2 (am): Remove media, replace with fresh media Day 4–5: Count fluorescent cells... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and... 75,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute 525,000 cells...

...FAM-CACTCCCTCTCTGCGCGCTCG-BBQ- Reagent Preparation 1X PCR Buffer containing 0.05% Pluronic F-68: Prepare immediately before...cool. Prepare the ITR master mix in a microcentrifuge tube as shown below. For 8 samples prepare enough...GC/mL, where GC is genome copies and generally represents the physical number of viral particles. Users...4475 µL Molecular Biology Grade Water Procedure Preparation Before handling any viruses, get materials ready...sufficient pipette tips and reagent reservoirs. Pro-Tip Prepare the viral dilutions in a separate biological safety...(dilution BSC) than that used for master mix preparation and droplet generation (droplet generation BSC...well plate sealer by gently touching the screen. Prepare the Serial Dilution Place a 48-well dilution plate...

...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. General Considerations For antibody-expressing...antibody-expressing plasmids containing the SV40 origin of replication use a HEK293 line stably expressing the SV40 ... containing the Epstein-Barr virus origin of replication use a HEK293 line stable expressing Epstein Barr...BCD TFX) to 37 °C. See below for other Reagent Preparation instructions. Wipe down all pipettes and equipment...equipment with 10% bleach prior to use. Reagent Preparation 100 mM Valproic Acid Dissolve 2.88 g valproic...Sterilize by passage through a sterile 0.22 µm filter. Prepare 10 mL aliquots and store the solution at -20 °C...

...prestained protein ladder on ice. Reagent Preparation Prepare IgG Standard Dilute the 2.5 mg/mL IgG isotype...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. Last Update: June 14, 2023 Workflow.... Add 8 µL of PBS to a total volume of 13 µL . Prepare the recombinant antibody Dilute or concentrate ...microfuge tube. Add 8 µL of PBS to total 13 µL . Prepare 1X MOPS buffer Dilute 25 mL of 20X MOPS buffer ...appropriate imaging system. Figure 1 Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa...Analyze . Select Gels . Select Select Next Lane . Repeat for all lanes. Plot the area under the curves for...

...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. General Considerations The ELISA...reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard and coat the plate Dilute ... antigen to determine the ideal concentration. Prepare serial dilutions of the purified antigen as follows...overnight at 4 °C . Section 2: Block the plate Prepare the wash buffer (0.05% Tween-20 in PBS) as follows...microplate shaker set at 400 rpm and shake for 1 min . Repeat steps 8–10 twice for a total of three washes. After...wash, aspirate the wash buffer from the wells. Prepare the blocking buffer (1% BSA in PBS) as follows:...

... virus on ice. Prepare the following dilution series of virus in DMEM complete: Prepare 7 microcentrifuge..., where TU is transducing units and generally represents the number of infectious viral particles. Users...Assay: RPP30, Human, Bio-Rad, 10031244 Reagent Preparation DMEM Complete: 500 mL DMEM, high glucose, pyruvate...µL of the first dilution into the next dilution. Repeat for all dilutions as indicated in the chart below...untransduced control. Pro-Tip For even seeding, prepare a batch for 10 wells with 3,000,000 cells in 13.5...concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples can be ...immediately or stored at -20 °C until ready to use. Preparing for ddPCR Thaw samples, primers/probe mixes, and...

...each step Reagent Preparation Master Mix: Count the number of samples (n) and prepare master mix for an... number of genome-containing particles in an AAV prep using qPCR, SYBR Green Technology, and Addgene’s... number of genome-containing particles of an AAV prep using SYBR green technology. This protocol was validated...vector cores. Our titers were similar to those reported by these institutions. This protocol is for a ...μL Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid stock of 2 x 10 9 molecules/μL to generate...plasmids to find a suitable one. Some labs have reported better results when the plasmid is linearized....min / 98 °C 15 sec / 58 °C 30 sec / read plate/ repeat 39x from step 3 / melt curve Example of plate set-up...

...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. Safety Warnings Sodium azide is...to room temperature (RT) before use. Reagent Preparation 1 M sodium phosphate monobasic monohydrate (NaH...column. Collect the flow through in a 250 mL bottle. Repeat steps 9–10 such that the supernatant passes through...Centrifuge at 1000 x g for 2 min to remove PBS. Repeat Steps 30–31 3x , discarding buffer from the collection...Discard the flow through into a waste container. Repeat steps 29-31 at least 4 additional times to ensure.... If the sample concentration is still too low, repeat the centrifugation until the volume of the sample...

... 2 (am): 18 h post-transfection. Remove media, replace with fresh media. Day 3-4 (am): Harvest virus Equipment...(PES) filter Transfection grade DNAs Reagent Preparation DMEM Complete (also known as D10): 10% v/v FBS...T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use cells that are below passage 15...Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Prepare a mixture of the 3 transfection plasmids into OptiPro...empirically determined. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using...following morning, carefully aspirate the media. Replace the media with 10 mL of DMEM Complete or OptiPro...

... Protocol: Kit-free Alkaline Lysis Plasmid Miniprep Prepare the following solutions: Solution I - Resuspension...you can find a protocol for kit-free plasmid mini-prep at the bottom of this page. Last Update: Feb. 8,...culture of bacteria . Pro-Tip Refer to appropriate DNA prep protocol for volume of bacteria to grow (low copy...pellet the bacteria before proceeding with DNA preparation. Pro-Tip If your entire overnight culture cannot... chloroform to the recovered aqueous DNA layer. Repeat steps 2-4. Note: Phenol-chloroform is a hazardous...

...Use this protocol to prepare LB agar plates with antibiotic in your lab....samples are sterilizing in the autoclave, you should prepare your plate pouring station: Find an empty section...antibiotics, and a section for active pouring. Prepare your antibiotics. Prior to adding your antibiotic...1000x stock solution. For example: If you’ll be preparing plates with a final concentration of 100 ug/mL..., dissolve by vortexing, and filter sterilize. Prepare a water bath at 60 ℃ with sufficient water to submerge...Only the Resistant Strain Grows If everything was prepared properly, you should see growth of only the resistant...

...conditioned medium prepared above to make a new cell solution at a concentration of 5 cells/mL. Prepare approximately...culture dish, Corning 430167 (optional) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-...well plate. Allow the cells to grow overnight. Replace the medium on the cells and collect the used (conditioned...a hemocytometer or other cell counter. Pro-Tip Prepare a relatively dilute cell solution (<10 6 cells/.... If you choose not to use conditioned medium, prepare this cell solution in fresh complete medium. This...