...TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning vector. The vector is 8901 base...-Cloning Vector I.2 Recipes I.3 Warranty information Back to Top A. pLKO.1-TRC Cloning Vector A.1 The ...use of this vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the...TRC-cloning vector. Addgene Plasmid Description pLKO.1 – TRC control Negative control vector containing... Tools page. Several other laboratories have deposited pLKO derived vectors that may also be useful for...TRC Cloning Vector The following steps were the prevailing guidelines when the pLKO.1 vector was created...Digest pLKO.1 TRC-cloning vector with AgeI. Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA...

...plasmid Cut vector - Background due to uncut vector Cut vector + Background due to vector re-circularization...ends on either side of the vector are compatible with each other, the vector is much more likely to ligate...fuse the two ends of the vector together). Protocol: Standard Insert + Vector DNA Ligation Before setting...amount of cut insert and vector to use for the ligation reaction. The volume of vector DNA and insert DNA used...where the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will work just fine. We ...opens in a new window) ligation calculator to easily quantify how much vector and insert DNA to use. Combine... ALWAYS do a vector alone + ligase control. This will allow you to verify that the vector was completely...

...of this tutorial we will discuss how to add new restriction sites to the MCS of an empty vector. However...purify vector While waiting for your oligos to arrive, conduct a restriction digest of 1μg of vector with...Mix the annealed oligos with cut vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard...insert of 50bp in length into a 5kb vector, mix 100ng of the vector with 0.75-6 ng of annealed oligos)....just as easily to add short tags or shRNAs to any vector (the procedure will simply differ in terms of primer...primer design). Let's assume that your favorite vector has a relatively limited MCS (BamHI - EcoRI - SalI...restriction sites and add them to your existing vector. It is a couple days of work that will pay off ...

...annealed but nicked vector product is then repaired during the replication cycle. Empty vectors for LIC typically...your PCR primers. Step 2: Linearize Vector In this example, the vector is cut at two points by the enzyme...subsequent reactions. Step 3: Create Vector Overhangs Treat the linearized vector with T4 DNA polymerase to "chew...treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing...allows for electrophoretic separation of linearized vector from the reaction mixture, and may provide counter-selection...shown here. The manufacturer of an LIC-specific vector will provide the homologous sequence which must...Search Addgene's collection of empty LIC cloning vectors Protocol Step 1: Design Your Primers Primer design...

...into sterile tubes. Store the tubes at -80 °C. After thawing, the solution can be stored at 4 °C for up to...before putting the CS5 in the incubator. Return the CS5 to the incubator for 96 h. This incubation time...Cells You may also like... AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference...Hemocytometer or cell counter Ice bucket CO 2 incubator Pipet controller Hazardous waste container pH ...pH meter Stir plate Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T cells (... glutaGRO 11 mL of 200 mM L-alanyl-L-glutamine. Store at 4 °C. D1 + 0.1 M sorbitol (optional) : DMEM ... solution may separate into phases. Aliquot and store at 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150...

... PPE protocols . Working in a BSL-2 laboratory requires laboratory glasses in addition to coat and gloves...also adhered to. None of the authors, contributors, administrators, or anyone else associated with Addgene... eat, drink, chew gum, or apply makeup in the laboratory. Before you start your experiment, make sure ...Only mechanical pipetting should be done in the laboratory. Don’t mouth pipette! There may be some times...officer. BSL-2 Guidelines Remember, the BSL-1 laboratory guidelines above are expected to be followed ...not be worn outside of the BSL-2 area. BSL-2 laboratories must be clearly marked as “BSL-2.” The names...names and contact information of the laboratory manager should be clearly visible in the room and on the ...

...personal protective equipment including laboratory coat, laboratory goggles, and gloves. Sodium azide containing...Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller Benchtop centrifuge compatible...tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 °C bead... sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to 1 month at 4 °C Adjust pH to 7.0 Autoclave... Autoclave or filter sterilize 1000X protease inhibitor cocktail 25 mg leupeptin 50 mg antipain 250 mg...Recombinant Antibody protocol ). Pro-Tip Add protease inhibitors to 1X if this has not already been done. We typically...of supernatant and add 250 µL of 100X protease inhibitor cocktail. Add 1 part Protein A/G binding buffer...

... are important for long-term storage of plasmids. Although you can store your plasmid DNA at -20°C, many...LB agar plate can be stored at 4°C for a few weeks. However, if you want to store bacteria for a longer...concentration of long-term glycerol storage is unknown. Most labs store bacteria in 15-25% glycerol. You ...for long-term storage. Protocols... Protocols Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids Creating...Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids You may also like... Bacterial Transformation...cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years. Protocol Video ...

...Equipment Biosafety Cabinet 4 °C Refrigerator Micropipettes and tips Pipettor and pipettes Benchtop centrifuge...conical tubes Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 °C bead...sterile 0.22 µm filter. Prepare 10 mL aliquots and store the solution at -20 °C. 1 mg/mL PEI-MAX Add 1 g ... through a 0.22 µm filter. Prepare aliquots and store at -20 °C until use. BCD TFX 1000 mL BalanCD HEK293...mL 200 mM Glutagro Do not add selective reagents Store at 4 °C until use. We suggest preparing fresh solutions...500 mL BalanCD HEK293 Feed 20 mL 200 mM Glutagro Store at 4 °C until use. We suggest preparing fresh solutions...solutions after one month. 1000X protease inhibitor cocktail 25 mg Leupeptin 50 mg Antipain 250 mg Benzamidine...

...transduce cells: Initial dilution factor of virus stock Dilution factor after additional 10-fold dilution...ddPCR) for lentivirus vector titration. Protocols... Protocols ddPCR Titration of Lentivirus Vectors ddPCR Titration of...of Lentivirus Vectors You may also like... Lentivirus Colony Formation Titration Assay Lentivirus Fluorescence...droplet digital PCR (ddPCR) to titer lentivirus vectors. This protocol specifically uses primers and probes...dilutions as indicated in the chart below. Dilution Factor Virus Volume to Dilute Media Volume Total Volume...fold dilution of the virus stock! Don’t forget to factor this into your calculations later (see calculation...

... gene of interest from an integrated lentiviral vector....Lines with Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral... gene of interest from an integrated lentiviral vector. Unlike the short-term protein expression observed...approaches, generating cell lines using lentiviral vectors enables long-term protein expression studies. Moreover...repeated transient transfection. Some lentiviral vectors deliver mammalian antibiotic resistance (e.g., ...lentiviral transgene. Note that not all lentiviral vectors deliver antibiotic resistance. This protocol was...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Reagents...

...will describe how to copy a cDNA from one vector into a new vector that is better suited for analyzing the...re-circularization of the vector. You should treat your digested recipient vector with a phosphatase prior...nearly any piece of DNA to be placed into a backbone vector of choice with minimal limitations. Background ...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-...manufacturer’s instructions. Isolate Your Insert and Vector by Gel Purification Run your digest DNA on an agarose...also a good idea to run an uncut sample of each vector to help with troubleshooting if your digests don...Once you have cut out and purified your insert and vector bands away from the gel, it is important to determine...

...sterile tubes. Store the tubes at -20 °C or -80 °C. After thawing, the solution can be stored at 4 °C for... the harvested media to a polypropylene storage tube and store at 4 °C between harvest. Centrifuge the...Lentivirus Production You may also like... Viral Vector Guides Mol Bio Protocols Introduction This protocol...be used to produce lentivirus from a lentiviral vector transfected into 293T cells using a polyethylenimine...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container pH ... of heat-inactivated FBS and 5 mL of glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS....45 μm PES filter. The viral supernatant can be stored at 4 °C for several hours but should be aliquoted...

...same orientation on your target vector. If you are not sure what vector to use, you can check out our Empty...expression vector. Using subcloning, you can easily move YGOI into a mammalian expression vector. Design ...be used to easily move any piece of DNA from one vector to another as long as it is already bounded by ...commonly used lab technique. For the purposes of this tutorial we will discuss how to move a cDNA from one plasmid...manufacturer’s instructions. Isolate your insert and vector by gel purification Run your digested DNA on an...of recovered DNA . Ligate your insert into your vector Conduct a DNA Ligation to fuse your insert to your... You should see two bands, one the size of your vector and one the size of your new insert. If you used...

...bacterial colonies. Equipment Shaking incubator at 37 °C Stationary incubator at 37 °C Water bath at 42 °C Ice...because bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly...for transforming plasmid DNA for the purposes of storage and amplification. Higher efficiency cells are ... (containing the appropriate antibiotic ) from storage at 4°C and let warm up to room temperature and ...and then (optional) incubate in 37°C incubator. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 ...approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid. Incubate the...antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min. Pro-Tip This outgrowth step allows...

...by Limiting Dilution You may also like... Viral Vector Guides Virus Blog Posts Addgene Protocols Viral... individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day 14–30: Analyze...pipette 200–1000 µL single channel pipette CO 2 incubator Pipet controller Hazardous waste container Microscope...heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Polyclonal stable cell pool: see our protocol...individual cultures due to lack of secreted growth factors, so not every cell type will be able to grow under... empirically, and that the cell population be monitored regularly for transgene expression. This protocol...using this protocol will vary based on several factors, including the growth properties of the cell lines...

...temperature, as prolonged storage in the cold room may damage it. Using a pipettor, transfer the top, aqueous...interphase layer, consider using a lower volume pipettor like a p200 to collect a majority of the aqueous...quantification , which can be modified for RNA. Store your RNA sample(s) at -80 °C to prevent RNA degradation...sample, consider making smaller aliquots of it and storing those in -80°C. Option #2 - TRIzol® Protocol Homogenize...DNA quantification , which can be modified for RNA.Store your RNA sample(s) at -80 °C to prevent RNA degradation...

...much virus as possible. Store at 4 °C for short term (2 weeks), or aliquot and store at -80 °C for long term... Ultracentrifugation You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Introduction... one (long) day. Equipment Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep (60% Iodixanol)...Sterilize by passing through a 0.22 μm filter and store at 4 °C. 1X PBS-MK buffer Dissolve 26.3 mg of MgCl...Sterilize by passing through a 0.22 μm filter and store at 4 °C. 0.1% Pluronic-F68 in PBS (A) Add 500 µL... Centrifuge at 350,000 x g for 90 min in a T70i rotor at 10 °C. Pro-Tip If you need more time, you can.... Carefully take the QuickSeal tubes out of the rotor and place them in a stable rack. **Make sure not...

...Protocols ddPCR Titration of AAV Vectors ddPCR Titration of AAV Vectors You may also like... AAV Titration...Titration by qPCR AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference Introduction...titer purified recombinant Adeno-associated viral vectors (AAV). This protocol specifically uses primers ...probes targeting the ITR elements in the viral vectors but can be modified for other targets. The dilution... Transfer the cartridge holder to the droplet generator. Close the lid and wait for the droplets to be...Reaction volume (20 µL) C = Copies/µL D = Dilution factor of virus V = Volume of virus in reaction mix (4...concentration obtained by ddPCR should decrease by a factor of 2 across the dilutions. In the example below...

... the two, use a conversion calculator or chart like the G Force calculator (Link opens in a new window...Introduction A centrifuge is a common piece of laboratory equipment used to separate different components...tabletop microcentrifuge with the lid open, the rotor with microfuge tubes in place, and the control panel...centrifuge, with tubes evenly distributed around the rotor. How to Centrifuge Now that you know the different...working smoothly. Place your sample tubes into the rotor so that they are distributed evenly around the center...sample. Place the centrifuge’s internal lid on the rotor (if it has one) and then close the external lid....