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  • Title: [An method for small hairpin RNA expression vector reconstruction for easy single restriction endonuclease identification].
    Author: Shan ZX, Lin QX, Fu YH, Deng CY, Yu XY.
    Journal: Nan Fang Yi Ke Da Xue Xue Bao; 2007 Sep; 27(9):1341-4. PubMed ID: 17884773.
    Abstract:
    OBJECTIVE: To develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis. METHODS: The double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis. RESULT AND CONCLUSION: DNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.
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