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No DNA Bands after PCR

No DNA Bands after PCR - PCR - Polymerase Chain Reaction Forum

No DNA Bands after PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 01-13-2012, 08:21 PM
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Default No DNA Bands after PCR



Hi all,

I need some advice/opinions.

Basically I did PCR to amplify some beef DNA, and then ran a gel a few times. Now the problem is last time I didnt get no band at all, and this time I see a slight faint band, the size of it is correct but it is hardly there and looks like a primer dimer.

The reagents i am using for PCR are:
Promega Master mix which includes 50 units/ml of Taq DNA polymerase supplied in a proprietary reaction buffer (pH 8.5), 400μM
dATP, 400μM dGTP, 400μM dCTP, 400μM dTTP, 3mM MgCl2

The quantity I am using is 12.5 ul.

Then, Dh20 6.5 in positive tube, and 7.5 in negative tube

My forward and reverse primers at 2.5ul for both tubes

and then lastly DNA in positive tube at 1.0ul

and we also use the mineral oil (50ul) for the PCR to stop evaporation etc

After, this I load 15ul of my samples on the gel (agarose gel is 1% made up of 0.5 agarose and 50ml 1xTBE) alongside the ladder. I use 5ul of the loading dye for each sample.

The DNA ladder comes out fine each time I do it.

Where am I going wrong???
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  #2  
Old 02-15-2012, 04:16 AM
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Default Re: No DNA Bands after PCR

Hi there

What is the size of your PCR product? Can you post a gel?

Sounds like you are not getting much in terms of reaction, most of the successful PCR's have very dense, bright bands after PCR (even loading just a microlitre from the reaction).

You may have primer dimers competing with the reaction, or just poor amplification conditions / DNA quality of template

Its hard to say unless you post a gel

cheers
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Old 05-03-2012, 03:23 AM
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Default Re: No DNA Bands after PCR

hi
can anyone help me..i am also not geting band after pcr.only 1 time i get a faint band but when i am repeating pcr again i am not geting any band..i use following
1.template 2 Ál
2.dNTP 1 Ál
3.Buffer 5 Ál
4.Taq 1 Ál
5.Primer 1 2 Ál
6.Primer 2 2Ál
7.water 38 Ál

i used annealing temp 68 ░ for 40 seconds
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Old 05-03-2012, 07:03 AM
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Default Re: No DNA Bands after PCR

where is your magnesium chloride?
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Old 05-03-2012, 07:35 AM
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Default Re: No DNA Bands after PCR

magnesium chloride is also added
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Old 05-04-2012, 07:51 AM
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Default Re: No DNA Bands after PCR

i am still waiting for any suggestion
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Old 05-07-2012, 03:55 AM
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Default Re: No DNA Bands after PCR

how do you prepare your PCR template? boiling? if yes prepare it fresh.

If possible do tell us roughly what sample are you using, high copy number or low copy number (mean is the DNA copy a lot in the sample? Plasmic DNA generally is high copy number, biological sample may differ sometime less.)

Did any of your friend use the PCR reagent for their amplifiction and does it work?
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