Help! PCR linearity

Dr. Duncan Clark duncan at genesys.demon.co.uk
Sat Mar 1 04:45:31 EST 1997


In article <33087357.620B at aa.wl.com>, Joanne Rogers <rogersj at aa.wl.com>
writes
>Hi there!!
>I need to know if anyone has a quick protocol on how to ensure one is 
>working within the linear range of their PCR machine.  I am currently 
>running a telomerase quantification assay and need, for validation, GLP 
>purposes, a quick way to ensure I am indeed within its linear range.
>If anyone has any ideas, I would really appreciate it.
>Thanks so much.
>Jackie
>PDRI

I don't know about quick but you need to either run a number of
identical PCR's taking one off every second cycle or so over the range
say 20 to 40 cycles. Run an aliqout on a gel and run image analysis on
that gel to get a plot of no. of cycles against band intensity. This is
only valid for that one target you are amplifying and will differ for
any other target. We have done this for QC purposes for the odd company
using a particular target to ensure that buffers, MgCl2 and enzyme etc
from different batches all behave the same. We have to be on the linear
part of the amplification curve to do this. 


Alternatively it can be done by using a Molecular Probes fluorescent dye
as described in Jan 1997 Analytical Biochemisty. I only saw this
yesterday and didn't make a copy. 

Duncan 

--------------------------------------------------------------------------------
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk



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