PCR smear in samples and negative control - (Jan/31/2006 )

I have a smear in all my bands, also in the negative control, I exchanged buffer, NTP mix, MgCl2, Ex Taq, orderd new primers and used new MQ autoclaved water, but the contamination is still there. If anyone has another idea why this is like that, I would be glad for any help.

thanks

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Where are you making your PCR mixes?

What's the origin of your template and your negative control? Is your negative control just water?

There's a fixed topic dealing with PCR contamination issues...

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You might need to clean your pipets I recommend to use special tips like the "Shaft guard" from Rainin there are well designed and avoid many troubles.

If you can disassemble your pipets use 10% H2O2 and rinse with EtOH 100% this will definetely remove any source of contamination on your pipets

Several recommandations
Set up the premix on a bench which wil NEVER see any AMPLIFIED PRODUCTS

Add your DNA in another place

NEVER bring back your amplified samles to the same place where you assemble your master mix

Pesji

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please forgive me if you think my question is irrelevant, but what is the size limits of your smear?...
I have a smear at around 200pb wich i think is due to primers (as there is a lot of them, migration may be affected...)

for testing the contaminations, i do a test in a neighbourg lab and i only pick with me the template and the primers...

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yeah, sounds lika primer smear to me.

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Your primers should be then very long cause if it's around 200bp it cannot be generate with primer dimer at least I'm not aware of that

Pesji

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