| |
| Name of Product | pAPL140-CMV-MAS1-zeo Lentiviral Particles |
| Catalog No | pAPL-140 |
| Gene | Human MAS1 |
| Promoter | CMV |
| Tag | |
Reporter |
| Resistance marker | Zeocine |
| Additional note | N/A |
| The Lentiviral Particles are produced from a standardized protocol using purified plasmid DNA, lentiviral packaging mix, and lipofectamin 3000. The protocol uses a third generation self-inactivating packaging system meeting BioSafety Level 2 requirements. The ready-to-use lentiviral particles are ideal for the transduction of a variety of mammalian cells including difficult-to-transfect, primary, stem, and non-dividing cells. |
| NOTE | The Lentiviral System used is a Third generation HIV-based, and the formed viruses are self-replication deficient. |
|
Provided as 1 vial of 1000ul of non-concentrated MAS1 lentiviral particles. It is sufficient for infecting 10 x 6 well plate of cells. Lentiviral particles are shipped on dry ice and must be stored at -80C upon receipt. Avoid repeated freeze-thaw cycles as this will reduce titers. |
|
The lentiviral expression construct was validated by full-length sequencing, restriction enzyme digestion and PCR-size validation using gene-specific and vector-specific primers. Product is confirmed free of bacteria, fungi and common Mycoplasma contamination. |
|
The following protocol is a general method for transducing adherent cells in six-well plate. Use it as a starting point for determining the optimal transduction conditions for your target cells. |
|
1. | Plate target cells in 2ml complete growth medium, 12-18 hr before transduction (Note. Use heat-inactivated FBS for transduction). |
|
2. | Prepare transduction medium. Add polybrene to 2 ml of complete growth medium to desired
concentration. The optimum final concentration of polybrene may be determined empirically but
generally falls within a range of 2-10ug/ml (Note. Polybrene is a polycation that reduces charge repulsion between the virus and the cellular membrane. Excessive exposure to polybrene (>24 hr) can be toxic to cells). |
|
3. | Thaw the 1 ml of the premade viruses when use it for the first time, Mix gently, but do not vortex. Divide the stock viruses into 100ul aliquot. Using 100ul of viruses each time for cells in each 6-well plate and keep the rest aliquots at -80C. (Note that each freeze-thaw cycle will decrease titer by 2 to 4-fold. Add proper volume of the lentiviral stocks into prepared virus transduction medium to obtain the desired MOI, the total volume of virus represents no more than 1/3 the final volume of prepared virus transduction medium). |
|
4. | Remove the plate(s) of target cells from cell culture incubator. Aspirate culture medium. Add prepared transduction medium with virus to the cells. (Optional) Centrifuge the cultures at 1,200 x g for 60-90 min at 32C or room temperature (Centrifugation can significantly increase infection efficiency). Incubate the plate(s) at 37C for 8 to 24 hr in a CO2 incubator. If you are concerned exposure to the polybrene, limit the transduction to 6 to 8 hr. |
|
5. | Remove and discard the virus-containing transduction medium and replace it with fresh growth medium. Continue to incubate the cells for 24 to 48 hr to allow the expressed protein to accumulate in the target cells. Harvest the cells for analysis or proceed with selection using the appropriate antibiotic. |
|
Biosafety Level | 2 |
|
Caution | Please strictly follow the rules of Handling BSL-2 materials. |
|
The viability of Angio-Proteomie's premade lentiviruses is warranted for 30 days from the date of shipment, and is valid only if the product is stored and cultured according to the information included on this product information sheet. |
