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it lyses the cells.
Lymphocytes from whole blood were separated by lysing the red blood cells (RBCs) using a hypotonic buffer (ammonium bicarbonate and ammonium chloride; Himedia) with minimal lysing effect on lymphocytes. Three volumes of RBC lysis buffer was added to blood sample and mixed by vortexing and inverting thoroughly for 5 min and centrifuged (Eppendorf 5415R) at 20,00 g for 10 min. The supernatant was mostly discarded, leaving behind ∼1 ml to prevent loss of cells. To the pellet, 3 vol RBC lysis buffer was added, and vortexing, inverting, and centrifuging steps were repeated two to three times until a clear supernatant and a clean white pellet were obtained. After the final wash, the supernatant was discarded completely, and the pellet was resuspended in 500 μl PBS, followed by addition of 400 μl cell lysis buffer (10 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl, 10% SDS, pH 7.5) and 10 μl proteinase K (10 mg/ml stock; Himedia).
The sample was vortexed to dissolve the pellet completely and incubated for 2 h at 56°C in a water bath (CW-30G; Jeio Tech) for lysis. An equal volume of phenol (equilibrated with Tris, pH 8) was subsequently added to the tube and mixed well by inverting for 1 min. The tube was centrifuged at 10,000 g (at 4°C) for 10 min, and the aqueous upper layer was transferred to a fresh tube containing equal volumes (1:1) of phenol and chloroform:isoamyl alcohol (24:1). The tube was mixed by inverting for 1 min and centrifuged for 10 min at 10,000 g (at 4°C).
SDS is an anionic detergent . It has 12 carbon long hydrophobic tail and an hydrophilic sulphate end. So this tail being non polar or hydrophobic enters the lipid bilayer component of the ruptured cells and the anionnic portion that is hydrophilic , then disrupts the organizationn the cell membrane thus solubilizing it. Hence on centrifugation these lipid can be removed. While on proteins it completely sorrounds and make it totally negative charge thus unfolding occurs. This unfolding proteins can then be easily hydrolysed by proteinase k. however SDS is active at 50 degree celsius for 2 hours or at 37 degree celscius left over night.