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Sex determination in domestic chicken (Gallus gallus)
Introduction
In sexually dimorphic species both the sexes can be distinguished by their phenotype. But the same idea cannot be used for monomorphic species. The domestic chicken Gallus gallus is sexually monomorphic. Male and female differentiation cannot be made in a monomorphic species merely by means of phenotypic attributes.
Heterogametic sex of a species is a condition where in which the sex chromosomes are not the same for male and female. The females of this particular species are heterogametic with sex chromosomes Z and W and males are homogametic (ZZ). In case of birds and reptiles the females would be having a Z chromosome and a W chromosome and males would be having two Z chromosome. For domestic chicken it’s very difficult to differentiate between males and females as it is a monomorphic species.
Molecular markers serve as important tools in identifying monomorphic species. The sex of domestic chicken can be identified by targeted amplification of chromo-helicase-DNA binding gene 1 (CHD 1). PCR amplified product can be run on a gel and the males can be identified by the presence of a single band and females show the presence of two bands.
DNA required for the study was isolated from muscles, blood and feathers. DNeasy Blood and Tissue kit (Qiagen, 2012) was used to isolate the DNA. Universal avian sexing primers developed by (Fridolfsson and Ellegren, 1999) were used for the present study.
The main aim of the experiment were to isolate the DNA successfully from muscles, blood and feathers and use the DNA for PCR reaction to distinguish between male and female of the domestic chicken. The experiment was based on previous experiment conducted by (Hogan et al, 2012). The experiment also intended to identify a fool proof method to isolate quality DNA from the various tissues mentioned above. The study was carried out to see the efficacy of identifying sex through universal primers targeting loci present on the sex chromosome and also to replicate the result successfully in other contexts as well.
Methods
DNA extraction and Visualization
DNA was extracted from blood, muscles and feathers. Qiagen DNeasy blood and tissue kit was used for isolation of DNA from blood tissues.
About 20 µl of Proteinase K was pipetted into a microcentrifuge tube and to this 166 µl of Phosphate Buffered Saline was added along with 4 µl of RNase A.
The chicken blood was then transferred to the tube and incubated at room temperature. This step was followed by the addition of Buffer AL. The whole mixture was then vortexed for 15 seconds and incubated at 56 ˚C for 30 minutes.
About 200 µl of 95% ethanol was added to the tube and mixed thoroughly.
The whole of the mixture was then transferred to DNeasy spin column and centrifuged at 8000 rpm for 1 minute. The flow through was discarded and the spin column with the bound DNA was placed into a fresh 2 ml collection tube and 500 µl buffer AW1 was added to the spin column, centrifuged at 800 rpm for 1 minute and flow through discarded.
The spin column was then placed into a new 2 ml collection tube, 500 µl buffer AW2 added, centrifuged at 14000 rpm for 3 minutes to dry column.
The column was then removed and placed into a new collection tube and centrifuged at maximum speed for 1 minute to remove residual ethanol present.
The column was then placed into a new 1.5 ml micro centrifuge tube and 100 µl of buffer AE pipetted into the centre of the membrane. The column was then incubated at room temperature for 1 minute and centrifuged at 8000 rpm for 1 minute. The eluted DNA was then stored in aliquots at -20 ˚C till further use.
Procedure for muscle tissue
About 20 g of muscle tissue was macerated using a sterile razor blade and transferred to a micro centrifuge tube.
About 180 µl of ATL buffer was added and vortexed for 15 seconds and 20 µl of Proteinase K was added, mixed thoroughly and incubated at 56 ˚C for 30 minutes.
To this mixture about 4µl RNase A was added mixed well by vortexing and incubated at room temperature for 2 minutes.
To the above mixture about 200µl of buffer AL was added mixed thoroughly by vortexing. Then about 200 µl of ethanol was added and mixed well by vortexing.
The mixture was then transferred to DNeasy mini spin column, centrifuged at 8000 rpm for 1 minute and the flow through was discarded.
The spin column was then put in a new collection tube and 500 µl of buffer AW1 was added and centrifuged at 8000 rpm for 1 minute and the flow through was discarded.
The spin column was transferred to a new 2 ml collection tube, buffer AW2 added and centrifuged at maximum speed for 3 minutes to dry the spin column.
The spin column was removed carefully and transferred to a new collection tube and centrifuged for...