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shRNA

Formats — Glycerol FAQs


MISSION shRNA


What should I do if I have trouble obtaining high yields of plasmid DNA?
How should I store my bacterial cultures?
What is the expected pDNA yield from glycerol stocks?
I have several of the MISSION shRNA clones in the bacterial stocks. I was wondering what the competent cells used are and if you have any information on the stability of the shRNA plasmids in these cells?


What should I do if I have trouble obtaining high yields of plasmid DNA?
Generally, high yields of DNA can be achieved with much of the collection; however, viral-based vectors are known to be somewhat difficult to prep. If particular clones are troublesome, we recommend streaking the bacterial stocks on LB/carbenicillin plates to isolate a single colony and DNA purification with GenElute™ Endotoxin free Midi (Product No. NA0200) or GenElute Endotoxin free HP Maxi (Product No. NA0400S) prep kits.


How should I store my bacterial cultures?
Glycerol stocks should be maintained at -80 °C. Cultures should be maintained fresh, avoiding prolonged storage at 4 °C.


What is the expected pDNA yield from glycerol stocks?
When clones are grown up under suggested conditions, the average pDNA concentration achieved is around 55 ng/µl with a range of concentrations from 20 to 120 ng/µl (50 µl elution). To achieve maximum yields, we suggest Sigma's GenElute HP Plasmid Miniprep Kit for single-prep purification, or the GenElute HP 96 Well Plasmid Miniprep Kit for high-throughput purification.


I have several of the MISSION shRNA clones in the bacterial stocks. I was wondering what the competent cells used are and if you have any information on the stability of the shRNA plasmids in these cells?
The E. coli strain is in DH5alphaT1R. Sigma sells the identical strain (i.e. identical genotype) called GC5. The TRC has published several studies on plasmid stability of the library in that strain (see "Genome-scale loss-of-function screening with a lentiviral RNAi library" and "A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen"). An early criteria when designing the plasmid vector was improved stability of the lentiviral sequences in E. coli over past retroviral/lentiviral libraries. That said, be aware that E. coli containing lentiviral plasmids do grow significantly slower when compared to bacterial strains containing standard cloning plasmids such as pUC18, pET vectors or pBR322. We routinely grow for 20–22 hours in TB mediums containing 100 µg/ml of carbenicillin (rather than ampicillin) as we see better stability using that antibiotic than we do with ampicillin. Harvest and process pDNAs immediately post-growth (do not let them incubate at 4 °C for hours or even days, as that will result in lower quality pDNA). We also have some preliminary studies that suggests that the shRNA clones passaged in such strains such as STBL-3 tend to grow more consistently and produce higher plasmid yields then DH5aT1R. We have not performed a larger, more comprehensive study to confirm this observation to date however... therefore use this information at your own risk.



Contact Us
For questions about the library, pricing and quotes or other concerns, please e-mail us at: MISSIONRNAi@sial.com.


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