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confirmation of my pcr product - (Feb/07/2006 )

i have done a pcr for my primer and i got my sequence and checked for its purity in gel. i have got a single band. when i again i did the pcr with the same product (above pcr product) as my template DNA . insted of a same single band ,i have got double bands near the same base pairs. i don't know how to make it
could you please help me?how could i get a single band? what will be the reason for that to happen?


wow that sounds kinda odd.

Was the original single band really thick? Maybe the the orignal band was really 2 bands close together. So when you gel extracted, you really got two differently sized products for the next PCR reaction. Maybe try a higher percent gel to make sure the original band was a single band if it was thick.

If it was clearly a single band originally...I really don't know.

Why are you using the PCR product as the DNA template anyways?


Hi meenakshi,

Do you mean you are reamplifying your PCR product? Dkao maybe right but maybe you are using too much template in your 2nd PCR, did you dilute your first amplification product before you reamplify?

The same question again (if you don't have problems in answering), why are you reamplifying your PCR product? I only use to reamplify if my 1st PCR product is not seen in agarose gel (for example, when amplifying paraffine purified DNA).