What restriction enzymes are recommended for diagnostic
digests of the clone DNA?
We recommend Pvu II. Restriction digest with Pvu II results in
three bands; however, clones with an additional Pvu II site in the
hairpin insert will produce four bands: 2513 bp, 2325 bp, 1478 bp and 776 bp.
The band sizes following digestion with Pvu II of the shRNA vectors are 3803 bp, 2513 bp, and 776 bp
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What is the method you recommend for screening the stable clones and is there a pair of primers that can be used for screening them by PCR?
Puromycin resistant clones may be screened via qRT-PCR using primers designed against PAC or the target gene of interest.
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.
The protein I intend to knockdown is very stable, so the expression of the siRNA must extend over a long period, where the cells are dividing.
Since the library is lentiviral based, it allows for stable integration of the shRNA into the host chromosome. Stable clones or populations may be selected via the addition of puromycin. Individual clones may be chosen and expanded. Due to the random integration of the shRNA, screening several clones allows for optimal knockdown of your gene of interest and verification of possible off-target expression patterns.
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For questions about the library, pricing and quotes or other concerns, please e-mail us at: RNAi@sial.com.
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