Preparation of crude cell extract for quantification of HIV reverse transcription products by PCR

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