shRNA

Clone Propagation & DNA Isolation

 
MISSION shRNA

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 HP Midi or Maxi prep kits.

 


Can the MISSION lentiviral particles be further propagated in the lab?
No, the viral particles cannot be propagated for biosafety reasons. The MISSION TRC lentiviral particles are replication incompetent. The particles are made using features of 2nd and 3rd generation lentiviral packaging systems. Genes for replication and structural proteins are absent in the packaged viral genome since these genes are supplied by other plasmids in the packaging cells. The viral genome contains only the region between the 5’ and 3’ LTRs of pLKO.1. In addition, the lentiviral vector contains a self-inactivating 3’ LTR that renders it unable to produce infectious virus once it integrates into the host chromosome.


Can I extract the vector directly from the lentiviral particle?
No, this is not possible. The vector is used to make the particles in the packaging or producer cells. The viral genome contains only the RNA version of the region found between the 5’ and 3’ LTRs of pLKO.1 (promoter, hairpin sequence, puromycin resistance gene, etc.)


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.


Your MISSION TRC shRNA clones are sequenced after cloning, but are they sequenced routinely after each round of amplification due to the high frequency of recombination that occurs in retroviral vectors?
We do sequence verify clones in a random fashion after copying. We sequence approximately 5% of clones of the last copy made. Our R&D group has not seen any evidence of recombination. The TRC library has been cloned into a backbone that was optimized to avoid these issues.


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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