Day 1:
Day 2:
(Note: buffers should be mixed and agitated before start.)
Day 3:
Day 4:
Day 5:
Reagents:
HBS 2X:
TE 1X:
CaCl2:
(Adapted from Trono Lab Protocols)
Troubleshooting:
Low vector titers are often caused by inefficient transfection of 293T cells. Transfection efficiency can vary significantly and depends on several factors including the following. (1) When the 293T cells are plated for transfection, they must be trypsinized well to prevent clumping and allow formation of a uniform monolayer. Cells should be 50% to 70% confluent at the time of transfection. (2) Plasmid DNA of high purity and quality is crucial to achieve high-efficiency transfection. DNA concentrationtion and quality should be determined by spectrophotometric analysis and gel electrophoresis. Commercially available plasmid DNA extraction kits can be used to obtain highly purified supercoiled plasmid DNA. Prior to transfection, the DNA should be sterilized by ethanol precipitation. The air-dried pellet is then resuspended in sterile deionized, distilled
water. (3) The pH of the 2× HeBS solution is particularly critical and should be exactly 7.05. It is important to bubble the 2× HeBS solution with air as the DNA/CaCl2 solution is added dropwise. Once added to the cells, a fine precipitate
should develop that is readily visible under the microscope.
Vector titers are also influenced by the stability of the vector particles, which is affected by factors such as temperature, pH, freeze-and thaw frequency, and incubation conditions. VSV-G-pseudotyped HIV-1 vector particles have a half-life of 10.4 ± 1.2 hr at 37°C, 1 to 2 days at room temperature, and about 1 week at 4°C (Higashikawa and Chang, 2001). Changes in pH can dramatically affect vector stability. Although VSV-G-pseudotyped HIV-1 vectors are stable at pH 7.0, their half-life at pH 6.0 or 8.0 is <10 min (Higashikawa and Chang, 2001). In
order to prevent pH changes in vector supernatants, add HEPES buffer to the 293T medium at a final concentration of 10 mM.
(Ref: Curent Protocols in molecular biology-Unit 16.22)
Concentration of Lentiviral Vectors