PCR product longer than template - why?
Posted 26 February 2009 - 05:01 AM
I got my 500bp product using the genome as template in my normal PCR buffer and conditions. After gel extraction, I used the 500 bp fragment as template with the following PCR conditions
A only the sense primer
B only the antisense primer
C both the sense and antisense primers
B has two similar bands 500bp,800bp ,but lighter. C has the 500bp ,same as the template.
So,how can you explain the results?
attached : the first lane on the right is A;the second lane on the right is B;the third on the right is C; the last lane is marker(1500,1000,900,800,700,600,500,400,……）
Posted 27 February 2009 - 04:50 AM
Posted 27 February 2009 - 07:21 AM
Perhaps you didnt get my meaning.In the second pcr ,I used the 500bp fragment as my template and then I got the 800bp with the antisense primer.
most likely this is because of non-specific primer binding to off-target sites in genomic DNA. Try to use software like (Oligo) to maximize primer design . But keep in mind that when the template genomic DNA it is sometimes difficult to fish your target especially if it was a gene promoter.
thank you so.