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Extrremely bright bands in the WELLs of PCR agarose gel


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8 replies to this topic

#1 WB9547

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Posted 27 February 2005 - 02:13 PM

Hello all,

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.

#2 vetticus3

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Posted 27 February 2005 - 05:35 PM

is your cDNA template from a plasmid? quite often if too much plasmid is used, it causes a band at a high level, never seen it in the well though. what is the concentration? too much template is never a good thing.

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.

#3 WB9547

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Posted 27 February 2005 - 07:05 PM

Hi Vetticus3,

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.

Thanks again.

#4 WB9547

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Posted 27 February 2005 - 07:08 PM

I forgot to mention that i am actually doing RT-PCR and hence my template is cDNA. Could there be any fault with my cDNAs?

#5 methylnick

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Posted 27 February 2005 - 07:21 PM

I think vetticus's point is you may have too much starting material for your RT-PCR. I suspect the band you are seeing at or near the well is in fact tcarried over from the template DNA for your reaction. You could try diluting the template DNA and see what happens then.

cheers

Nick

#6 pcrman

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Posted 28 February 2005 - 02:33 AM

Another possibility is that your sample got stuck to the well and didn't migrate at all. Try to use fresh made gel and fresh buffer.

#7 seqgirl

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Posted 28 February 2005 - 06:44 AM

See above:
Pinned: smearing on a gel.

#8 WB9547

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Posted 28 February 2005 - 02:51 PM

Thank you all for your replies. My problem is solved after i changed the Taq from Pfx (invitrogen) to Taq - Qiagen. I dont know the reason behind this. Thats the only change i made this time. I did a PCR today and everything worked fine. I didnt see any well in the gel stuck with PCR product. Thank you again.

cheers.

#9 Great White Northern

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Posted 05 March 2005 - 08:43 AM

That seems like a bit of an unlikely solution. Once, the glycerol used to make our loading buffer got contaminated and grew bacteria/mould. The DNA in the PCR product/plasmid formed complexes with proteins and other microbiotic junk, and smeared down all the way from the wells.
In your last experiment, did you use fresh loading buffer?




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