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