Nested PCR - (Sep/05/2007 )

Hi,

I was trying some nested PCR. The first PCR volume is 15 microlitre. Then I am diluting the primary PCR 1:500 times and taking 1 microlitre for the secondary PCR. Although I am getting the desired band I am also getting a big smear. Why is it so.

Thanks

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hi,
i had similar problems,i set a 25 microlitre reaction mix,took 1 microlitr,diluted 1:100,from which i took 1 microlitre for next round ,it came well.
in ur case,try more dilution till the smear goes,thats what i did,
thanks,best of luck

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Thanks for the reply. I tried different dilutions, however it is again a smear with the band. Anyone has any other suggestions.
Thanks

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What for do you need the nested PCR product??

If you just want to clone it and if you have the right band, just cut it as fine as you can.

Can you see the first PCR product??
May be alignment temperature is too low.

I have the same problem sometimes; most of them get fixed diluting the first PCR product.
You could try cleaning with a PCR purification kit before the second PCR.

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Run the first PCR on a gel, stick a pipet tip into the correct band, and swish the pipet into the second PCR tube.

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Thanks for the reply. I want to purify the product and sequence it.
In some cases the first PCR product is visisble and in some not visible. However in all the second product is visible with the smear. I am trying to reduce the taq units and in some cases the smear is gone. Some are still having the smear. I am doing lot of samples (cDNA) at a time. I don't think that it is a dilution problem. There are multiple problems and each sample needs to be dealt seperately.

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