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Int J Mol Sci. 2011 Jan 18;12(1):613-26. doi: 10.3390/ijms12010613.

Construction and characterization of a cDNA library from wheat infected with Fusarium graminearum Fg 2.

Author information

1
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada; E-Mails: khaledta72@hotmail.com (K.A.-T.); anita_brulebabel@umanitoba.ca (A.B.-B.).

Abstract

Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART) technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

KEYWORDS:

15 acetyl deoxynivalenol (15ADON); 3 acetyl deoxynivalenol (3ADON); Fusarium graminearum Fg2; Triticum aestivum; cDNA library

PMID:
21340003
PMCID:
PMC3039969
DOI:
10.3390/ijms12010613
[Indexed for MEDLINE]
Free PMC Article
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