Do you guarantee knockdown using your clones?
Yes. Based upon the design algorithm developed by The Broad Institute, at least one clone per target set (set of 4-5 clones) should yield greater than 70% knockdown of the targeted transcript. Validation of the library is underway. Generally more than one clone in each set gives significant knockdown. Proper controls and conditions are required.
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Figure 1. Silencing of 40 different gene targets. MISSION shRNA lentiviral particles were used to transduce several human cell lines. Percent gene expression was determined by measuring mRNA transcript levels and comparing to the MISSION pLKO.1-puro Control Transduction Particles (SHC001V). Knockdown of all individual lentiviral constructs was greater than 70%.
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Which cell lines or types have been tested?
We have a list of cell types that have been successfully transduced by pLKO.1-puro based lentiviral particles.
After selection with puromycin, why are there no surving cells?
The cells you are working with may be sensitive to puromycin. The concentration of puromycin added to the cells may be too high. For each new cell type used, it is recommended that a puromycin titration be performed to determine the lowest concentration of puromycin needed to efficiently select transfected or transduced cells.
Puromycin titration (kill curve)
- Plate 1.6 x 104 cells into wells of a 96-well plate with 120 ml fresh media.
- The next day add 500 – 10,000 ng/ml of puromycin to selected wells.
- Examine viability every 2 days.
- Culture for 10 – 14 days. Replace the media containing puromycin every 3 days.
- The minimum concentration of puromycin that causes complete cell death after 3-5 days should be used for that cell type.
Contact Us
For questions about the library, pricing and quotes or other concerns, please e-mail us at: MISSIONRNAi@sial.com.
MISSION is a registered trademark of Sigma-Aldrich Co. LLC Label License.
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