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Identification of putative odorant receptors from Epiphyas postvittana 90 4.2.10 Genome Sequencing For genomic sequencing of E. postvittana, the genomic DNA (gDNA) was extracted from purified nuclei. This was done to reduce the amount of mitochondrial DNA (mtDNA) being sequenced. The nuclei was isolated first before gDNA extraction as outlined in section 4.2.11. Library construction (using the Paired-End DNA sample prep kit VI from Illumina), cluster generation and genomic analysis was carried out at the Allan Wilson Centre for Genome Sequence (AWCGS, Palmerston North). A total of 8 lanes of 75 bp paired-end genome sequencing were done on the Illumina 1G Genome Analyzer (Illumina) at AWCGS. Pipeline analysis was done at the Bioinformatics department at Plant and Food Research Ltd by Ross Crowhurst. The sequence data was assembled using SOAPdenovo assembler (Li et al., 2008; Li et al., 2009). Resulting scaffolds were deposited into the genome server held at Plant and Food Research Ltd. The putative OR sequences identified from the transcriptome data, together with known B. mori OR sequences (Krieger et al., 2005; Nakagawa et al., 2005; Wanner et al., 2007) were used for searching the genomic scaffolds for their E. postivttana homologues using tblastn. Scaffold hits with e-value less than 0.5 were taken as significant. 4.2.11 Nuclear DNA isolation and mitochondrial DNA contamination test [This protocol has been modified from the method by (Guo; Lopez-Gomez and Gomez-Lim., 1992)]. 4.2.11.1 Isolation of insect nuclei Refer to Appendix C for buffer recipes used in this section. Three grams of female E. postvittana pupae were ground in a mortar and pestle in liquid nitrogen and transferred to a beaker with 300 mL of ice-cold nucleic extraction (NEB) complete buffer. The resulting homogenate was filtered through 4-6 layers of cheesecloth into a sterile glass beaker on ice. The filtration step was repeated through 2-4 layers of miracloth into a 500 mL sterile glass cylinder. The volume was adjusted to 294 mL with NEB complete buffer and 6 mL of 25% Triton X-100 in NEB complete buffer. The cylinder was sealed with parafilm and mixed very gently by inversion 10-20
Identification of putative odorant receptors from Epiphyas postvittana 91 times. The homogenate was divided into six 50 mL Falcon tubes and spun down at 588 rpm at 4-10°C, for 2 minutes. The supernatant was transferred to a new set of 50 mL Falcon tubes and centrifuged at 3300 rpm, 4-10°C for 15 minutes. A reddish pellet was clearly visible. The supernatant was transferred to the waste container and each pellet resuspended with 50 mL NEB– no -mercaptoethanol and mixed gently by inversion until the pellet was resuspended. The samples were spun down as before and the supernatant transferred to the waste container. Each pellet was resuspended with 5 mL NEB– no -mercaptoethanol and all pellets collected into one weighed Falcon tube. More NEB– no -mercaptoethanol was added to a final volume of 50 mL, spun down again as before and the supernatant transferred to waste container. The tube was weighed to find out the total amount of nuclei isolated and the tube kept on ice. 4.2.11.2 Genomic DNA extraction 0.2 grams of nuclei pellet was resuspended in 14 mL lysis buffer and mixed by inversion. 14 µL of RNase A 50 mg/mL (Qiagen) was added and mixed by inversion, followed by incubation at 37°C with gentle shaking for 10 minutes. 1.4 mL 5 M potassium acetate pH 7 was then added to it followed by addition of 3.5 mL 100% ethanol. The sample was vortexed at maximum speed for 30 seconds and extracted with equal volume of chloroform:isoamyl alcohol (24:1). The tube was then placed horizontally in a platform shaker at room temperature and shook gently for 5 minutes. The sample was spun down at 3000 rpm for 10 minutes at room temperature and the supernatant collected and placed in a new 50 mL Falcon tube. Equal volume of ice cold isopropanol was added to the sample and mixed by inversion, followed by incubation at -20°C for 30 minutes. The sample was spun down as before, the pellet washed with same volume of 70% ethanol. The DNA pellet was air dried at room temperature for 20-30 minutes and resuspended in 100–200 µL TE buffer pH 7.5. The DNA quality was checked at OD260/280 on the nanodrop (Thermo Scientific). 4.2.11.3 Mitochondrial DNA contamination test A ten-fold serial dilution of the gDNA was made from 1 in 10 to 1 in 100 million with TE buffer. The primer pairs MT6 forward 5‟- GGAGGATTTGGAAATTGATTAGTTCC-3‟ and MT9 reverse 5‟-
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
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Contents 4.2.5 Degenerate PCR .....
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List of Figures List of Figures Fig
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List of Tables List of Tables Table
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List of Abbreviations gDNA Genomic
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1.1 General overview 1 General Intr
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General Introduction 3 moth in loca
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General Introduction 5 Figure 1.1:
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General Introduction 7 1.5 Componen
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General Introduction 9 OBPs have al
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General Introduction 11 A model has
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General Introduction 15 Tanaka et a
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General Introduction 17 Table 1.1:
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General Introduction 21 et al., 200
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General Introduction 23 the attract
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General Introduction 25 leaves and
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General Introduction 29 Expression
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General Introduction 31 BmOr2 HvOr2
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General Introduction 33 for lepidop
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Functional characterisation of Epip
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Functional characterisation of Epip
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Page 109 and 110: Microarray OR 454 Transcriptome OR
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Page 149 and 150: Concluding Discussion 133 sequencin
Page 151 and 152: Appendix B 135 Buffered TB (Sambroo
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Appendix E 141 I II
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Appendix E 143 V
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Appendix E 145 VII continued Figure
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References 147 Ansebo, L., Ignell,
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References 149 Butenandt, A., Beckm
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References 151 Fedurco, M., Romieu,
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References 153 Große-Wilde, E., St
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References 155 Hrdy, I., F. Kuldova
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References 157 Kaissling, K. E. (19
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References 159 Krieger, J., Große-
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References 161 Lopez-Gomez, R. and
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References 163 Miura, N., Nakagawa,
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References 165 Pell, J. K., Macaula
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References 167 Røstelien, T., Stra
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References 169 Stowers, L. and Loga
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References 171 Turner, C. T., Davy,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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