Bio 594 Research Paper Sample

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Princy Mary Paulose Bio 594 Research paper 05-25-2011 In-situ hybridization of tau mRNA in transgenic Drosophila melanogaster third instar larvae Abstract: The purpose of this research was to study the transcription of human tau DNA in Drosophila melanogaster third instar larvae, and to detect the presence of tau mRNA wherever it was expressed. Drosophila flies transgenic for human tau were mated with flies having specific tissue-specific promoters which directed tau expression to specific tissues in the progeny. The promoter lines have specific GAL-4 transcription factors, which binds to UAS enhancer sequence upstream of tau promoter in the transgenic flies,…show more content…
The purpose of these primers were to select specific regions that will be amplified. Using primers TauT7F1 (5’ - ATC CCA GAA GGA ACC ACA GCT GAA - 3’) and TauT7R1 (5’ - TGT TTG GTC AAC TGG ACT CGT TCC - 3’), PCR reactions (based on standard PCR protocols) were employed to amplify the tau DNA region in the plasmid. 25 μL of master mix with the plasmid template DNA was treated with 1 μL each of forward and reverse primers, and 25 μL of water was finally added to make it to a total volume of 50 μL. Spectrometric analysis and agarose gel electrophoresis were conducted to analyze purity of the PCR product. From the gel electrophoresis, we found that the PCR product was about 600 bps. Spectrometric analysis helped us to find the concentration of amplified DNA which was about 65 μL/mL. The next step was to produce digoxigenin-UTP labelled RNA probe by in vitro transcription of PCR amplified tau DNA with T7 RNA polymerase. About 4 μL of template DNA was added to a sterile, RNase-free reaction vial. The total sample volume was made up to 13 μL by adding water. The reaction vial was placed on ice, and was added 2 μL each of 10x NTP labeling mixture, 10x transcription buffer, and T7 RNA polymerase. 1μL of protector RNase inhibitor was also added, and the contents in the vial were mixed gently and incubated for 2 hours at 37 degree Celsius. To remove the template DNA after transcription, 2 μL of DNase I was added and incubated for about 15 minutes at 37 degree Celsius. The reaction was stopped by adding 2 μL of 0.2 M EDTA at pH 8. Probe RNA was precipitated by centrifuging at 14,000 rpm at 4 degree Celsius for thirty minutes. The RNA transcripts were then analyzed by spectrometric and gel electrophoresis techniques. Spectrometric results showed an absorbance of 0.371 A at a wave length of 257 nm. We then calculated the concentration of RNA as 14.84 ng/μL, which

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