Sexual differentiation in monomorphic avian species
Madeleine Evans ID: 213023921, Deakin University Waurn Ponds, Course S323 Biomedical Science, Subject SLE254 Genetic
Introduction
Sexual differentiation is one of the first biological processes to occur during the development of a foetus. There are a variety of methods for sex determination amongst vertebrates including analysis of primary and secondary differentiations. Primary differentiation refers to the presence of gonads, while secondary looks at the phenotypical differences that occur in dimorphic species. Although, not all species are dimorphic (Klug W.S. 2014). Many of the avian species are monomorphic, meaning that there is no obvious differences in appearance between male and female …show more content…
Directions for the nano drop was run accordingly to machine directions. For electrophoresis, 10 µl of the sample was transferred into a clean microfuge tube with 2 µl of 6x loading dye. The portion of the sample was pipetted into an agarose gel and run at 120V for roughly 60minutes. Upon completion the gel was photographed for conformation.
PCR Preparation
A master mix was prepared in accordance to the PCR set up table provided by laboratory demonstrators resulting in a positive female, positive male and a negative control tubes. 40 µL of the PCR master mix was pipetted into a 0.2mL PCR tube. 10 µL of the prepared DNA solution was added the tube and placed in the thermocycler. A program optimised to amplify CHD1W and CHD1Z gene variants with the 2550F -2718R primer set was run.
Final Electrophoresis
An agarose gel was prepared and left to set. 10 µl of prepared DNA was pipetted unto a new microfuge tube with 2 µl of 6x loading dye. The solution was pipetted into the prepared gel next to a molecular weight and positive and negative controls. The gel was run with a high voltage buffer at 300V for 20 minutes. The gel was removed and visualised using the Gel Doc system.
Gel Doc photos where analysed, comparing the DNA samples with the controls to determine sex and molecular weight of PCR …show more content…
If DNA is not stored correctly, the results obtained could be considered inaccurate or insufficient. Degradation of DNA refers to the breakdown of the molecule into smaller fragments which can occur when exposed to environmental factors, thus why non-invasive methods of DNA accumulation can be unreliable. Feathers, faeces or skin collected from the wild are often left in the open and subjected to heat and sunlight, fracturing the regions of the DNA molecule (Wong, Wiley et al. 2012). DNA must be stored away from sunlight, temperature and pH extremes as well as environments that have high concentrations of salt. Muscle tissue should be kept at -80°C when stored in liquid nitrogen, or frozen at -25°C. This is the same for whole blood tissue samples including that of feathers(Seutin, White et al. 1991). Other factors contributing to the deterioration and result inaccuracies include time of storage and contamination of
Madeleine Evans ID: 213023921, Deakin University Waurn Ponds, Course S323 Biomedical Science, Subject SLE254 Genetic
Introduction
Sexual differentiation is one of the first biological processes to occur during the development of a foetus. There are a variety of methods for sex determination amongst vertebrates including analysis of primary and secondary differentiations. Primary differentiation refers to the presence of gonads, while secondary looks at the phenotypical differences that occur in dimorphic species. Although, not all species are dimorphic (Klug W.S. 2014). Many of the avian species are monomorphic, meaning that there is no obvious differences in appearance between male and female …show more content…
Directions for the nano drop was run accordingly to machine directions. For electrophoresis, 10 µl of the sample was transferred into a clean microfuge tube with 2 µl of 6x loading dye. The portion of the sample was pipetted into an agarose gel and run at 120V for roughly 60minutes. Upon completion the gel was photographed for conformation.
PCR Preparation
A master mix was prepared in accordance to the PCR set up table provided by laboratory demonstrators resulting in a positive female, positive male and a negative control tubes. 40 µL of the PCR master mix was pipetted into a 0.2mL PCR tube. 10 µL of the prepared DNA solution was added the tube and placed in the thermocycler. A program optimised to amplify CHD1W and CHD1Z gene variants with the 2550F -2718R primer set was run.
Final Electrophoresis
An agarose gel was prepared and left to set. 10 µl of prepared DNA was pipetted unto a new microfuge tube with 2 µl of 6x loading dye. The solution was pipetted into the prepared gel next to a molecular weight and positive and negative controls. The gel was run with a high voltage buffer at 300V for 20 minutes. The gel was removed and visualised using the Gel Doc system.
Gel Doc photos where analysed, comparing the DNA samples with the controls to determine sex and molecular weight of PCR …show more content…
If DNA is not stored correctly, the results obtained could be considered inaccurate or insufficient. Degradation of DNA refers to the breakdown of the molecule into smaller fragments which can occur when exposed to environmental factors, thus why non-invasive methods of DNA accumulation can be unreliable. Feathers, faeces or skin collected from the wild are often left in the open and subjected to heat and sunlight, fracturing the regions of the DNA molecule (Wong, Wiley et al. 2012). DNA must be stored away from sunlight, temperature and pH extremes as well as environments that have high concentrations of salt. Muscle tissue should be kept at -80°C when stored in liquid nitrogen, or frozen at -25°C. This is the same for whole blood tissue samples including that of feathers(Seutin, White et al. 1991). Other factors contributing to the deterioration and result inaccuracies include time of storage and contamination of