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Non specific amplification in PCR

Non specific amplification in PCR - Molecular Biology Techniques

Non specific amplification in PCR - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 03-01-2012, 06:12 AM
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Angry Non specific amplification in PCR



I am doing PCR with single stranded template in two steps. In first step a primer is used with 15 extra nucleotides at 5' end and 18 nucleotide complimentry with template (total 33 nt long). this primer can extend over single stranded template. when there is enough amount of complimentry strand of template then there are two more primers, one designed for 15nt overhang and other for second end of this amplified strand so long primer of 33nt is key primer and without addition of it there must be no amplification but i am getting problem that every time i run PCR and put all reagent except long primer as negative control, there is amplification of righ size product. i took each n every precaution to avoid contamination so much so to resynthesized all sequences two times and set PCR very far from my lab area with different aliquote each time. but all in vain. can anyone help me????
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