Lysis buffer Composition:
1M Tris-HCl pH 8.0……………...100ul
0.5M EDTA……………………….20ul
3M NaCl………………………….330ul
10% SDS…………………………500ul
Sterile water upto……...........……..10ml
Procedure:
• Take single mosquito in an eppendorf tube and add 50ul lysis buffer.
• Homogenize in Ice
• Add equal volume of Phenol
• Centrifuge at 8000rpm for 10 mins
• Take the supernatant
• Add equal volume of Chloroform-Isoamylalchohol(24:1) mixture
• Centrifuge at 8000 rpm for 10 mins and take the supernatant
• Add 1/10th volume of 3M sodium acetate
• Add 2.5 volume of chilled Ethanol
• Keep at -20°c overnight
• Centrifuge at 10,000rpm for 5 mins
• Decant the supernatant
• Wash with 70% alchohol
• Centrifuge at 10,000rpm for 5min
• Decant the supernatant
• Air dry the pellet
• Dissolve the pellet in 50ul of sterile water.