Extrremely bright bands in the WELLs of PCR agarose gel
Posted 27 February 2005 - 02:13 PM
After running the gel at 80V for 40 minutes, I am seeing really sharp and bright bands in the wells of my PCR gel. A smear is following that bright band from the well all the way to the end of the lane. In some lanes loaded with same samples, i am seeing the band of interest at the exact molecular weight i want, but still i see these sharp bands in the wells even in these lanes too. i am not seeing this band in the marker loaded well. Can any one give any suggestion how to over come this problem? In my PCR i use high levels of Mg+2..is this the cause?
What those bands signify? Is higher or lower concentrations of any chemical can cause this? Should i add anything in the PCR mix.
I use following recepie for PCR:
10X amplification buffer (invitrogen) 2 microl
MgSo4 (2.5 mM invitrogen) 3 microl
10mM dNTP 1 microl
Primer F 1 microl
Primer R 1 microl
cDNA 1.5 microl
Taq pfx (50mM invitrogen) 0.4 microl
ddH20 10.1 microl
Please give me any sort of advise.
thanks in advance.
Posted 27 February 2005 - 05:35 PM
do you see the band in your negative control?
most likely it's contamination.
high levels of Mg usually cause nonspecific amplification. but if you're not getting a lot of bands that mean sweet FA, then don't worry about it.
wouldn't think that it would cause what your seeing.
Posted 27 February 2005 - 07:05 PM
thanks for the reply. I am not using plasmids. My RNA is from lung tissue samples. I dont think it is a contamination problem. Because the problem happens once in a while and not every time i run the gel. When it goes right, I get no background with bands of interest lying exactly at the same position as i want. No additional bands whatsoever. But this problem appears in the other times. That means the chances are 50:50. As far as i know, i am consistent with the reagents and the protocol i use for all the good and bad experiments. I am unable to understand what might be causing the problem. How would teh PCR product get stuck up in the well? I am still getting my band of interest when the product is stuck in the well, but not that intense as i would get otherwise. This means my product is also stuck in the well. What could possibily stop the flow of PCR product through the gel? and also iam seeing this problem in negative controls too.
Posted 27 February 2005 - 07:08 PM
Posted 27 February 2005 - 07:21 PM
Posted 28 February 2005 - 02:33 AM
Posted 28 February 2005 - 02:51 PM
Posted 05 March 2005 - 08:43 AM
In your last experiment, did you use fresh loading buffer?