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...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% Poloxamer 188 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...