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Smears on PCR products


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4 replies to this topic

#1 OA17

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Posted 10 May 2010 - 02:05 AM

Hello!

I guess smears appearing when you run a PCR, is a common theme on PCR discussions, and it is certainly something that usually happens to me. However, I still don't know what is the exact reason, at least in my case. I have uploaded a photo of my gel (agarose 0.8%) in which I have run an aliquot of my PCR products.
1: Molecular size marker.
2: PCR-A
3: PCR-B
4: Negative control (No DNA template, but the rest of the master mix is the same as in the case of lanes 2 and 3)

Why do I get the smears on lanes 2 and 3, if the PCR seems to have worked correctly ? (I have obtained the expected bands!). It seems to be specific of the DNA templates, because in the case of the negative control, where I put water instead of DNA, I don't see this smear....

Any explanation for this?

Thanks in advance!

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  • 100508pcrsM.jpg


#2 criscastells

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Posted 10 May 2010 - 02:35 AM

Hello!

I guess smears appearing when you run a PCR, is a common theme on PCR discussions, and it is certainly something that usually happens to me. However, I still don't know what is the exact reason, at least in my case. I have uploaded a photo of my gel (agarose 0.8%) in which I have run an aliquot of my PCR products.
1: Molecular size marker.
2: PCR-A
3: PCR-B
4: Negative control (No DNA template, but the rest of the master mix is the same as in the case of lanes 2 and 3)

Why do I get the smears on lanes 2 and 3, if the PCR seems to have worked correctly ? (I have obtained the expected bands!). It seems to be specific of the DNA templates, because in the case of the negative control, where I put water instead of DNA, I don't see this smear....

Any explanation for this?

Thanks in advance!


How many DNA did you use to perform PCR program? And which volume of the PCR product did you load in the agarose gel?

#3 Adrian K

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Posted 10 May 2010 - 09:59 AM

From what I see from the GEL picture, based on my experience you had put too much DNA template into your PCR master mix. I dont have my gel in hand at the moment to show you this, but this happens to me when I put too much of template. This is frequently happen to me when I do colony pcr where I had put too much of colony into it.

Just my 2 cents.
Adrian

Edited by adrian kohsf, 10 May 2010 - 10:01 AM.

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#4 Maddie

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Posted 12 May 2010 - 05:42 AM

I also believe you used too much DNA. If it's due to unspecific amplifications though, just play with the MgCl2 concentration.
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#5 OA17

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Posted 13 May 2010 - 07:37 AM

Ok, thanks to all!

It is possible that I have used too much DNA for this PCR.
However, when I try to do reamplifications using Pwo polymerase, I also get the smear on the negative control! (which does not have DNA but water instead)... and that makes me think of some kind of problem with the reaction mix... I don't know.... sounds strange...

Thank you!




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