...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...

...able to start an overnight culture for plasmid DNA prep the following day. Tips and FAQ The optimal concentration...glycerol stock before you begin your plasmid mini-prep. Try not to freeze/thaw your glycerol stock too ...15-25% glycerol. You can prepare the glycerol stock the same time you prepare your plasmid DNA. In the...

... do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might want...inoculate bacteria in liquid culture. Protocol Prepare liquid LB. For example, to make 400 mL of LB, weigh...

...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...Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep (60% Iodixanol) QuickSeal tubes QuickSeal tube spacers...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...Centrifugal filter units (MWCO 100 kDa) Reagent Preparation 1 M NaCl/PBS-MK buffer Dissolve 5.84 g of NaCl... and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation and loading of the iodixanol gradient: 15% iodixanol...

...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... 75,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute 525,000 cells...thaw the lentiviral aliquot on ice prior to use. Prepare dilutions of the lentivirus into DMEM complete ...

...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... Use this dose for the colony formation assay. Prepare a batch of DMEM complete containing 10 μg/mL polybrene...thaw the lentiviral aliquot on ice prior to use. Prepare 10-fold serial dilutions of the lentivirus into...

...NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal. Close the lid and click ...

...polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously filtered) Microcentrifuge...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 (...determine the lowest dose that kills all of the cells. Prepare a batch of DMEM complete + 10 µg/mL polybrene by...Thaw the lentiviral aliquot on ice prior to use. Prepare a range of dilutions of the lentivirus in DMEM ...cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute 350,000 cells...growing well and have been sufficiently expanded, prepare cell stocks and/or harvest to test for protein ...

...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... Protocol: Kit-free Alkaline Lysis Plasmid Miniprep Prepare the following solutions: Solution I - Resuspension...pellet the bacteria before proceeding with DNA preparation. Pro-Tip If your entire overnight culture cannot...

...appropriate imaging system. Figure 1 Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa... Pro-Tip Addgene only uses recombinant antibody preps that have a purity of ≥90% but some labs use unpurified...prestained protein ladder on ice. Reagent Preparation Prepare IgG Standard Dilute the 2.5 mg/mL IgG isotype.... 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 ...

... 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...each step Reagent Preparation Master Mix: Count the number of samples (n) and prepare master mix for an...μL Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid stock of 2 x 10 9 molecules/μL to generate...

...plate. Be sure to pick multiple colonies for mini-prepping and verify insert by sequencing. Reference Page...

... °C for 45 min. You are now ready to purify your prep . The clarified supernatant can be kept overnight...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...

...Conical tubes (Fisher, 14-432-22) Reagent Preparation Prepare, sterilize, and pour all LB Agar + Antibiotic...representation is maintained. Maxipreps - Less is more: Do not overload the Maxipreps as yield can dramatically... high efficiency (1x10 10 cfu/µg) commercially prepared electrocompetent cells is strongly recommended...Antibiotic 65 mm Petri dish (VWR, 11019-552) 4 MaxiPreps (Qiagen HiSpeed Max, Catalog #12663) Tips (1000...access to sterile scraper and cold LB. Pro-Tip Prepare one to two more conical tubes on ice in case you...spread out the harvested cells further than four Maxipreps worth. Count colonies on the most dilute Petri.... It will be close enough for the purpose of a Maxiprep. Purify plasmid DNA using the Qiagen HiSpeed Maxi...

...determination. Prepare a 50:1 Reagent A to Reagent B dilution of the BCA assay. Prepare serial dilutions...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...instructions for your specific tank. Close the clamp. Prepare 1X running buffer as follows: Dilute 25 mL of 20X...for 15 min in 20% ethanol in deionized water. To prepare 20% ethanol, dilute 2 mL of ethanol into 8 mL of...

...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...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...well freezer block and place in the dilution BSC. Prepare 1X dilution buffer (see recipe above). Save ~25...

... virus on ice. Prepare the following dilution series of virus in DMEM complete: Prepare 7 microcentrifuge...Assay: RPP30, Human, Bio-Rad, 10031244 Reagent Preparation DMEM Complete: 500 mL DMEM, high glucose, pyruvate...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... plate sealer by gently touching the screen. Preparation of the Master Mix Place a ddPCR plate onto a ...freezer block and set aside in the BSC to cool. Prepare the RRE/RPP30 master mix in a microcentrifuge tube...

...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...Recommended Materials Material Vendor and catalog # DNA Miniprep Kit Qiagen #27104 EcoRI NEB #R0101S NcoI NEB #...based on your experiment Detailed protocols for preparing polybrene, protamine sulfate, and puromycin are... BL2 safety practices should be followed when preparing and handling lentiviral particles. Personal protective... 5g yeast extract 10g sodium chloride 15g agar Prepare LB agar solution by dissolving 40g of LB powder...store at 4°C. Hexadimethrine Bromide (Polybrene) Prepare a 1mg/mL solution of polybrene (Sigma-Aldrich catalog...

...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... Filter the solution through a 0.22 µm filter. Prepare aliquots and store at -20 °C until use. BCD TFX... reagents Store at 4 °C until use. We suggest preparing fresh solutions after one month. BCD Feed 500 ... Glutagro Store at 4 °C until use. We suggest preparing fresh solutions after one month. 1000X protease...

...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...