After running my PCR on a gel, I got double bands showing for all the positive samples. However, the expected band was brighter than the other band I observed. The cycling conditions were 94 C- 5 min, 40 cycles (94 C- 1 min, 50 C- 1 min, 72 C- 1 min), 72 C- 1 min, 4 C.
If the band is faint, it could be contamination, if it's bright (though not as bright as the expected band) it could be there is similar binding site at other location other than the region that you are aiming. In another case, if your positive sample refers to positive control using lambda dna, then, it's most probably contamination. So, what you can do is:
1) if it's the first reason, try amplify from different tube of dna and try do avoid contamination
2) if it's the second reason, meaning that no matter how many times you extract the dna an amplify, you still have double band, then you may have to consider designing a new set of primers that are more specific to your target region.
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