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Author: Wu, H
Author: Mo, Y
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Methods Article

Generation of shRNAs from randomized oligonucleotides

Hailong Wu1, Anh Dinh1 and Yin-Yuan Mo1*

1 Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine. Springfield, IL 62794. USA.

* To whom correspondence should be addressed: Yin-Yuan Mo, Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University. 801 N. Rutledge PO Box 19626, Springfield, IL 62794. USA. Phone: 217-545-8505. Fax: 217-545-7569. Email: ymo@siumed.edu

Biol. Proced. Online 2007;9:9-17. doi:10.1251/bpo129
Submitted: February 15, 2006; Accepted: March 28, 2007; Published: July 04, 2007.

Indexing terms: RNA Interference; RNA, Small Interfering; Oligonucleotides.

Abbreviations: bp, base pair; RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, short interfering RNA.


Figure 2 Enlarged

Fig. 2:

Generation of p53-shRNA. The same strategy described in Fig. 1 was used to construct p53-shRNA. A: PCR and Bcc I digestion. PCR amplification yielded a 150 bp band (lane 1) and digestion of the PCR product with Bcc I shifted the band to 130 bp (lane 2). B: Ligation. Bcc I-digested DNA fragment was ligated to siRNA-loop-1 at 4°C overnight. Lane 3, before ligation; lane 4, after ligation. C: Extension. Ligated DNA fragment was treated with the nicking enzyme N.Alw I and then extended by Taq polymerase in the presence of dNTPs. Lanes 5, before nicking; 6, after nicking; 7, after extension. The extended product was about 160 bp. D: Cloning. Plasmid DNA was isolated from five positive clones and then digested with BamH I and Kpn I. M, 25 bp DNA ladders. All gels are 2%.

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