Extra Bands

[gene26]

when i did a nested PCR (Mouse BAC) i got the bands near expected 3745bp but i dont know what are those high intense bands below 1.5kb

what could be the reason of getting those bands..??? even in nested PCR

image.jpg

[relaxin]

Since I do not know what your primers are specific to, I just guess. It is possible that the nested primers share sequence homology with some non-target region (repetitive sequence) or high GC region. See if you can eliminate the extra band by raising the annealing temperature. Redesigning the nested primers may be an alternative.

[mchlbrmn]

If you did too many cycles in the second stage of the nested PCR, then that's a secondary/nonspecific band from the second primers. With nested PCR you get the boosted specificity of two primer sets only if the cycle number is restricted enough so that the second primer set cannot produce products without the boost from the first PCR reaction.

What Relaxin said is good advice in either case, and that advice of redesigning primers or raising anneal temp can solve the possible problems both of us mention. If the PCR is sufficiently specific, the extra cycles won't matter. Actually, given that the smaller one is more intense one of these solutions may be necesary (unless the big product plateaued at a lower intensity, and the small product only came up later).