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How to interpret this agarose gel?

PCR PRIMER AGAROSE GEL

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6 replies to this topic

#1 thinker

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Posted 10 February 2012 - 07:37 AM

Hello everybody,

Someone could help me to interpret this gel?
I did a PCR to confirm the efficacy of my recently designed primer. The primer must to product a 127pb amplicon. I see a band with this size in the samples 4 in both kits. But something appears below 100pb. What is this? Why my primer works in sample 4 and in the others not?

Some sugestions?

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#2 Rosario

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Posted 10 February 2012 - 08:27 AM

This bands probably are the primer dimers products that can occur during the PCR because the annealing of primers each others.
Usually they do not compromise the PCR results and the forward experiments because are lost after purification of PCR product.
You could try to use less primers concentration, but in my opinion is not a big problem.

Which are the differences between the reactions that you loaded in the different lanes?

Bye,
RV

#3 thinker

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Posted 10 February 2012 - 09:01 AM

This bands probably are the primer dimers products that can occur during the PCR because the annealing of primers each others.
Usually they do not compromise the PCR results and the forward experiments because are lost after purification of PCR product.
You could try to use less primers concentration, but in my opinion is not a big problem.

Which are the differences between the reactions that you loaded in the different lanes?

Bye,
RV


Hi RV,

But do you think that my primers are bad designed? I did not see any secundary structures with in silico tests.

Actually the samples are simple controls. They are from different animals but with no treatment. I did this PCR just to test my primers. I used two different kits that we already have in my lab just to test if they have the same efficacy.

I could not understand these differences!

#4 scolix

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Posted 10 February 2012 - 11:14 AM

Its difficult to explain primer behavior. They have a mind of their own.
But I agree lane 4 seems more specific and others probably primer dimers

#5 Trof

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Posted 10 February 2012 - 01:31 PM

I agree with thinker and scolix, just wanted to add I would go for mix X. There seems to be specific amplification in line 4, and very faint in line 2. When there is no acceptable template, dimers are preferentially amplified. But line 4 seems OK with X mix, no dimers. Maybe the others are simply negative (of faintly positive in case of 2) and dimers are non-template dimers. Try more positive controls to see if you get dimers in samples.
Of course in Taqman system dimers should not be detected by probe, but may decrease the efficiency of reaction.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

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#6 thinker

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Posted 11 February 2012 - 02:49 AM

Hi there,
Thank you for your help.
So, the primers were bad designed. I did test my primers for secundary structures and they showed low delta G for dimers (-3.61 kcal/mole for self-dimer and -4.74 kcal/mole for Hetero-dimer), so I think that were good primers, but maybe the Cs and Gs at the end of them (CGATGTTAGAGAAGCCCAC, GCTATCACCAAGTCCTCC) facilitate the dimers results. Am I wrong?
Also, the quantification of cDNA of all samples was 714, 500, 692 and 775 ng/uL, respectively. So, I think that they have similiar templates. Am I wrong again?

#7 Trof

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Posted 11 February 2012 - 08:56 AM

Both primers have high 3' stability. One of them has 4 G/Cs in last 5 bases, that's a lot. And both primers have a low Tm.That could cause strong binding of the 3' part, while the rest of them doesn't bind.
Maybe the problem is not the stability of dimers itself.
Your cDNA can have similar concentration but different levels of your gene. It's hard to say.
I would redesign the primers. Choose ones with less stable 3' end and higher Tm. When you only care about the product on gel, you can do with messy primers as long as they're sufficient for your needs. But If you want to quantify, the primers better be good.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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