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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 1 Enlarged

Fig. 1:

Illustration of cloning strategy. A: Incorporation of 19 randomized nucleotides into PCR products. PCR reactions were performed using the cloned H1 promoter as a template and primers H1-5.1 and H1-R-3.2. B: Ligation of siRNA-loop-1. After PCR, the PCR product was digested with Bcc I and then ligated to siRNA-loop-1. C: Generation of palindromic sequences. Digestion with the nicking enzyme N.Alw I generated a partially single-stranded and double-stranded structure. At 72C Taq polymerase converted the single-stranded DNA to double stranded DNA. D: Cloning. The extended product was digested with BamH I and then ligated to pSK-H1-Pu-X that had been digested with BamH I and Xcm I.

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