novel approaches to expression and detection of oestrus in dairy cows

3.2.4 Sequencing of DNA in the Laboratory
Four blood samples were sequenced in the laboratory undergoing DNA
extraction, polymerase chain reaction (PCR) amplification, gel extraction
for purification and then sequencing of the gondaotrophin releasing
hormone receptor (GnRH-R) gene. The GnRH-R gene was chosen for
sequencing in the laboratory for a learning exercise because primers for
this gene had been previously optimised in the laboratory.
3.2.4.1 DNA Purification
Frozen samples were incubated at 37˚C (Mini 18L CLAD Incubator) and
defrosted rapidly to aid red blood cell lysis. 3ml of blood was then purified
using the Gentra Puregene Blood Kit (Qiagen, Sussex, UK). The of blood
was added to 50ml tubes containing 9ml of Red Blood Cell (RBC) Lysis
Solution, supplied with the kit, vortexed and centrifuged (DuPont Sorvall
RC5C) at 10000rpm for 15 minutes. The supernatant was removed, the
pellet resuspended in 9ml RBC solution and the process repeated twice
more, to ensure ample lysis of cells. After final centrifugation, the
supernatant was discarded, the pellet resuspended in the remaining
residual and 3ml of Cell Lysis Solution (supplied with the kit) added to
dissolve all remaining structures into solution, aided by incubation at 37˚C
(Mini 18L CLAD Incubator) for 1 hour.
Post incubation samples were cooled on ice to aid precipitation of the
proteins. 50ml chloroform and isoamylalcohol (both sourced from Sigma-
Aldrich, Dorset, UK) was prepared (49:1) and 3ml added to 50ml phase
lock gel tubes (Eppendorf, Stevenage, UK), along with 3ml phenol (10mM
Tris HCl, pH 8.0, 1mM EDTA; sourced from Sigma-Aldrich, Dorset, UK).
DNA in the Cell Lysis Solution (from previous step) was transferred to the
phase lock gel tubes, vortexed and centrifuged (DuPont Sorvall RC5C) at
10000rpm for 15 minutes. Using the phase separation technique allowed
differentiation between the DNA and the protein. DNA in the upper aqueous
phase was separated from the protein fraction in the lower organic phase.
The DNA sample was transferred to clean 50ml phase lock gel tubes and
phase separation carried out again using 5ml chloroform isoamylalcohol.
After the second centrifugation the DNA aqueous layer was transferred to a
new 50ml tube containing 1ml of Protein Precipitation Solution (supplied
with the kit) and centrifuged again for 15 minutes.
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