Preparation of crude cell extract

for quantification of HIV Reverse Transcription products by PCR

Prepare Crude extracts:

• Transfer 100,000 cells in a 1.5ml tube (Starstedt)

• Pellet the cells at 5,000 rpm for 2 min

• Remove all the supernatant

• Resuspend in 100µl of K buffer supplemented with 100 µg/ml of proteinase K (molecular biology grade (nuclease-free) PK from      Boehringer).

• Vortex

• Incubated 2 h at  56˚C

• Spin down liquid briefly

• Inactivate the proteinase K by incubation 10 min at 95˚C to inactivate the proteinase K

• Perform reaction with 10ul of extracts into 100ul of total volume of PCR

K buffer : 50 mM Tris-Hcl (pH8.3), 50 mM KCl, 1.5 mM MgCl2, 0.5% Tween 20, 0.5% Nonidet P40