×
Please leave your comments or questions
 
Preparation of crude cell extract for quantification of HIV reverse transcription products by PCR
Author: Marielle Cavrois, updated date: , view: 30, Q&A: 0
Tags: HIV and Productive Infection

Procedure

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

 


Comment
Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.