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Smearing in nested pcr product - (Feb/28/2006 )

Currently i am doing a nested pcr in mycobacterial DNA. First i am amplifying 396bp product and from which i take 1 microlitre and perform another pcr to get a 225 bp product.
after pcr,when gel is done i can see the first pcr product is nice and clear,while the nested product comes as smears,with light bands at the 225bp region.
I have been increasing the annealing temp, Mg conc is 1.5mm, Annealing time was reduced but the same result,
Kiindly suggest some way to avoid this smear and to getr a nice band,
Thank you


If you can easily visualise your first pcr product, dilute it before using it as template for the second pcr. Try 1/10 to 1/100 and use 1 ┬Ál.


Thank you,i will try that