Virus Packaging Technology 2 - Lentivirus Production and Operation Manual

Ruisai Small Class 0625-107
First, the experimental process
The lentiviral expression plasmid and its helper vector were prepared. Four plasmids were co-transfected into 293T cells, and replaced with complete medium at 6h after transfection. After 48 and 72 hours of culture, the supernatants of the cells rich in lentiviral particles were collected. The supernatant was concentrated by ultracentrifugation.

Second, the experimental materials
The lentiviral vector packaging system is a four- plasmid system consisting of Gag/pol, vsvg, rev and pLenti-X-EGFP expression vectors. Polo3000 transfection reagent, etc.

Third, the culture of packaging cells 293T cells
(a) Cryopreservation of 293T cells
As the number of passages increases, 293T cells will have decreased growth status, mutations, and the like. In order to prevent such phenomena from appearing, we need to freeze the cells in large quantities at the beginning to ensure the stability and sustainability of the experiment. Cryopreservation in the logarithmic growth phase increases the survival rate of cell resuscitation.
1. Remove the supernatant and add PBS to wash away the remaining medium.
2, was added 0.0 5% trypsin, digestive half 1 min, were observed when cell rounding, increased intercellular space, trypsin is removed, fresh medium was added and mix by pipetting, transferred to a centrifuge tube.
3. Count the cells, shake all the cells, add 2 mL of pre-warmed 10% DMEM at 37 °C, blow with a 10 mL pipette, blow 6-8 times with a large force, leave no dead ends, and then, all cells Aspirate, place in a 15 mL centrifuge tube, take 50 ul of the mixed cells in a 1.5 mL eppendorf tube, add 450 ul of 10% DMEM, then dilute 10 times, mix, and take 10 ul of cells in a counting plate. There are 4 large grids on the counting board, 16 small grids per large grid. When counting, the 4 large cells are counted, the total is divided by 4 (the number of cells per cell), and multiplied by 10 (10-fold dilution), which is the actual n-m / mL cell concentration.
4. Centrifuge the cells at 1000 rpm for 5 min. Remove the supernatant.
5. Add cell cryopreservation solution (70% complete medium + 20% FBS + 10% DMSO) according to the cell count result, and resuspend the cells at a density of 4 x 106 cells/ml.
6. Pack into the cell cryotube, place it in a freezer box, and put it into a -80 °C ultra-low temperature freezer.
7. Place the cells in a liquid nitrogen tank for a long time to store.
(B) the passage of 293T cells
When the cells grow to a confluence rate of 80% to 90%, the cells need to be subcultured to expand the number of cells and maintain a good growth state of the cells.
1. Digest the cells, and the method is frozen with the cells.
2. After the cells are centrifuged, resuspend in complete medium.
3. According to the specific conditions, the cells were divided into 10 cm culture dishes, and each culture dish was supplemented to 10 ml of medium.

4. Place the culture dish back into the incubator at 37 ° C and 5% CO 2 .
(C) 293T cell resuscitation
When the number of cell passages is too high, the state of the cells is deteriorated, or when the cells are contaminated, it is necessary to discard and resuscitate the cells that were originally frozen.
1. Set a water bath with a temperature of 37 °C.
2, check the cell bank record, according to the record, remove the frozen cells from the liquid nitrogen tank (need to wear cotton gloves to prevent frostbite), quickly throw into the water bath and shake quickly, try to dissolve the cell solution in 1min. .
3. Transfer the cell solution to a 5 ml centrifuge tube and add 1 ml of fresh complete medium to it, mix and centrifuge, 1000 rpm, 5 min.
4. Remove the supernatant, add 2 ml of fresh complete medium, mix the pellets, and transfer to a 6 cm culture dish.
5. Place the culture dish in an incubator at 37 ° C, 5% CO 2 and 95% relative humidity.
6. Observe cell viability on the second day. Change the cells for the medium. Cell growth was observed daily afterwards.

Fourth, lentivirus packaging and concentration
( a ) 293T cell culture
The cells were adjusted to the appropriate state and plasmid co-transfection was performed at a density of about 70%. Replace the cell culture medium with antibiotic-free medium before transfection.

( 2 ) Transfection
The transfection reagent Polo3000 was mixed with the minimal medium (please refer to the Polo3000 reagent manual), the plasmid DNA was mixed with the minimal medium, and after incubation for 5 min at room temperature, the two were mixed and incubated in a 37-degree incubator for 15 min. Then, the mixture was added to a cell culture dish, mixed, and the cells were placed in an incubator to continue the culture. Change the fresh medium 6h after transfection.
(4) Virus collection
The virus supernatant was collected twice at 48 and 72 h after transfection , collected and filtered through a 0.45 μm filter, and centrifuged at 72000 g/min for 120 minutes at 4 °C.
(5) Virus resuspension and preservation
The virus was resuspended in 1 ml of PBS and stored in a 4 degree refrigerator for one week. If it needs to be stored for a long time, it should be stored at -80 °C.
Five, infected cells
Different cells have different MOIs. Before the virus infects the target cells, pre-experiment is needed to determine the optimal MOI of the cells and whether the auxiliary enhancer Polybrene needs to be added.
(a) cell preparation
Inoculate the well-preferred cells into a 24-well plate to a cell concentration of 2 × 10 5 /ml. The number of cells inoculated is slightly different depending on the growth rate of the cells, which is generally to ensure cell confluence when the virus is infected the next day. rate of 50%.
(two) infection
a group of added polybrene ,   The other group does not add. Within MOI = 10 to 50, add 2 wells per MOI value , add a group of infection enhancer polybrene to make the final concentration of polybrene 8 ug/ml, and change the solution after 24 hours. (Refer to the attached table for the number of viruses to be added)
(C) Observe the fluorescence expression after 48 hours, determine the MOI value of the cells and whether Polybrene needs to be added.

Potassium Tert-butoxide CAS No.865-47-4

Potassium tert-butoxide Basic Information
CAS: 865-47-4
MF: C4H9KO
MW: 112.21
EINECS: 212-740-3

Mol File: 865-47-4.mol

Potassium Tert-butoxide Structure

density 0.910 g/mL at 20 °C

Potassium Tert-butoxide Application

1. Used in pesticides, medicines, printing and dyeing, Catalysts, etc.
2. As a strong base, it is widely used in the condensation, rearrangement and ring opening reactions in organic synthesis such as chemical, pharmaceutical and pesticide.
3. It is a moderately strong base commonly used in organic synthesis.
4. The reason why t-BuOK is widely used is that it is inexpensive and readily available, and its basicity changes depending on the selected reaction solvent.
5. For Stobbe condensation, t-BuOK is a better base than EtONa, with higher reaction yield, shorter reaction time, and no side reactions of ketone or aldehyde reduction.

Potassium Tert-Butoxide,Potassium Tert-Butoxide Cas No,Potassium Tert-Butoxide Msds,Potassium Tert Butoxide Formula

Shandong YingLang Chemical Co.,Ltd , https://www.sdylhgtrade.com