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Theses

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2006 - 20102
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Subject: Biotechnology and Molecular Biology × Author: Supriya × Type: Thesis ×
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    ThesisItemOpen Access
    Genetic Diversity Analysis And Qtl Mapping In Pearl Millet (Pennisetum Glaucum) Using Diversity Arrays Technology (Dart)
    (Chaudhary Charan Singh Haryana Agricultural University; Hisar, 2010) Supriya; Yadav, R.C.
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    ThesisItemOpen Access
    Isolation of drought related MYB transcription factor gene from brassica carinata
    (CCSHAU, 2006) Supriya; Yadav, Neelam R.
    The present study was conducted to isolate the drought related MYB transcription factor gene from Brassica carinata using RT-PCR approach. Primers were designed using AtMyb2 gene sequence of Arabidopsis thaliana, a well established drought induced Myb gene. No similar sequence was found in Brassica species when gene sequence was used in BLASTN search using NCBI website www.ncbi.nlm.nih.gov. The primers were based on conserved DNA binding domain of Myb gene. Brassica carinata seeds were grown on MS medium for 14 days, seedlings were uprooted and then exposed to air for 1hr/2hrs.dehydration treatment. Total RNA was isolated using Trizol Reagent method. Total RNA yield varied from 2250-2262.5 μg/g fresh wt. and two distinct ribosomal bands (28S and 18S) were observed when total RNA was analysed on formaldehyde and agarose gels. Isolation of mRNA was done using Genelute mRNA miniprep kit and its concentration ranged from 148.8-211.7μg/g fresh wt. i. e. one-tenth of the total RNA. RT-PCR was carried out using Qiagen one step RT-PCR kit with Q-solution and total RNA as well as mRNA as template. All the three sets of primers specific to Myb gene showed amplification. Set III (GhMYB) amplified a product of around 200 bp. A common band of approx. 500 bp was observed with Set I and Set II with mRNA as template. The cDNA was eluted from gel and further amplified. The amplified fragment was transformed using pPCR ScriptTM Amp SK(+) Cloning Vector (Stratagene) and pDrive cloning Vector (Qiagen) in XL-Blue strain of E. coli using blue-white selection. Transformation frequency in the range of 10.00 to 21.05 per cent was observed. The transformed clones were characterized by carrying out plasmid DNA isolation, PCR amplification of plasmid DNA and restriction enzyme digestion of plasmid DNA. The isolated plasmid DNA from transformed (white) colonies showed higher vector size when analyzed on agarose gel suggesting that DNA fragment was inserted into vector. Further, the plasmid DNA restriction pattern confirmed the cloning event.