... Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection...This procedure can be modified for alternative packaging cell lines or transfection reagents. Once produced...PES) filter Transfection grade DNAs: Lentiviral Packaging plasmid(s), Envelope plasmid, and Transfer plasmid...Considerations Before You Start The health of the packaging cell line is critical for obtaining high viral... 15 for viral production. Procedure Seed 293T packaging cells at 3.8×10 6 cells per plate in DMEM Complete...into OptiPro SFM: Reagent Amount per 10 cm dish* Packaging Plasmid ( psPAX2 ) 1.3 pmol Envelope Plasmid (...supernatant at 2100 rcf for 5 minutes to pellet any packaging cells that were collected during harvesting. Filter...

...columns have a finite binding capacity. If your sample exceeds the capacity, divide the sample among multiple...columns. For information about the total binding capacity for your columns, refer to the manufacturer’s ...Place a mark on the side of the column where the compacted resin is slanted upward. Place column in centrifuge...centrifugation step Pro-Tip Resin will appear compacted after centrifugation. Discard the flow through.... Slowly apply the sample to the center of the compact resin bed. Pro-Tip For sample volumes <1.5 mL top...the reservoir of the column. Fill the remaining space in the column with PBS and pipette several times...through into a waste container. Fill the remaining space in the column with PBS and pipette several times...

...control vector containing scrambled shRNA. psPAX2 Packaging plasmid for producing viral particles. pMD2.G ...particles. Note: pLKO.1 can also be used with packaging plasmid pCMV-dR8.2 dvpr and envelope plasmid pCMV-VSVG...tubes VWR: #87003-290 Note: pLKO.1 could also be packaged using pCMV-dR8.2 dvpr and pCMV-VSVG from the Robert...transfection: 1 μg pLKO.1 shRNA plasmid 750 ng psPAX2 packaging plasmid 250 ng pMD2.G envelope plasmid to 20 μl... may want to vary the ratio of shRNA plasmid, packaging plasmid, and envelope plasmid to obtain the ratio...

...you want to expand that with the addition of NdeI, PacI, AscI, and MfeI. With oligo overlap cloning, you...the original vector. Designing oligos To add NdeI, PacI, AscI and MfeI sites between the EcoRI and SalI ...the additional sites in tandem ( NdeI - CATATG , PacI - TTAATTAA , AscI - GGCGCGCC , MfeI - CAATTG ). ...

...Procedure The balance in the photo above has a capacity of 200 g, as indicated by the number above the...is best. Check that the balance has the correct capacity for the material that you are weighing by looking... you’re weighing out an amount larger than the capacity of your balance or weighing boat, you can weigh...

...experiment, make sure your workspace is clean and uncluttered. Disinfect your workspace with appropriate materials...times for each agent. Make sure you have enough space to work at your lab bench. It’s possible that while...

... differences, and plasmid recombination can all impact the representation of individual plasmids in the...problem as these sequences are not efficiently packaged into lentiviral particles. See below for options...overloaded. If you find that your pellet outstrips the capacity listed in the Maxiprep protocol, scale the reagent...

... appropriate for the biosafety level of the lab space Reagents Sample to be centrifuged Water Background...picture above. Wear PPE appropriate for the lab space in which you are working. Even if your samples may...

...remain in the laboratory. When traveling between lab spaces, take your glove off of one hand and use your bare...

...at a lower voltage. You will want to have enough space around each band to cut without having DNA in other...

...bath, and be sure to know what the maximum fill capacity is. Look for a “fill” line inside the water bath...

...personal protective equipment, setting up a clean workspace, and maintaining sterility while working. AAV ...

...to the amount you are dispensing can negatively impact your experimental results. This protocol will guide...

...(60% Iodixanol) QuickSeal tubes QuickSeal tube spacers 16 ga needle 18 ga needle 10 mL syringe 18 ga blunt...

... important to have nice crisp bands and to have space to cut out the bands. Because of this we recommend...

... important to have nice crisp bands and to have space to cut out the bands. Because of this we recommend...

...gel as follows: Remove the gel from the plastic packaging. Remove tape and plastic comb. Rinse the wells...

...hazards for these reagents. Work in a well-ventilated space and under a fume hood when working with the volatile...

...degrade any residual DNA carried over from the packaging process. Incubate at 37 °C for 45 min. You are...