RAPD Problems - Control lanes with bands

[Michael Cooley]

ANTHONY WILSON (002233w at axe.acadiau.ca) wrote:


:    I've been performing RAPD reactions using primers from the UBC lab 
: and have been running a control with the same stock as my tests (Primer, 
: taq, buffer, dNTPs, H20) replacing the DNA with sterilized water, yet I am 
: consistently getting banding patterns in these lanes WITHOUT DNA...  I have 
: replaced the H20, dNTPs, taq and buffer and yet the bands are as strong as 
: ever!  Is this a common setup for a control lane?


:    These banding patterns differ from reaction to reaction and from primer 
: to primer, yet bands appear in control lanes even when some of the test 
: lanes are clean...  Has anyone had any similar experiences and/or any 
: suggestions as to where in the protocol this error might be introduced??


:    Any help would be greatly appreciated.


: Thanks...

: Tony Wilson

The problem is that DNA is contaminating your TAQ. It is in all the TAQ 
polymerase sources at some level and is generally not a problem. The 
level is generally low enough that when template DNA is added the "real" 
bands are preferentially amplified. Later, Mike