I am conducting hemi-nested One-Step PCR assay on rectal swabs from bats stored in RNAlater. In the first 300 samples, I got clear and beautiful gel. Recently, the gel picture became as the attached image. This was only after running the second PCR . I changed the primers, reagents, diluted the samples, but nothing succeeded.
Please, have any of you experienced such situation? and what I should do?
How long it has been stored. The figure seemingly suggests the degradation of the sample. Have you repeated an earlier sample to see if the issue is related to sample or reagents?
I think you have post pcr product contamination of one of your reagents and it is amplifying hugely and the product is annealing with itself and forming longer products. Run a no template control sample which should be negative ( No bands at all) and if anything amplifies when you have no template dna then you have contamination
What is your expected amplicon size? There is no band for the positive control. This suggests that some thing went wrong. Please check all your reagents and standardize your PCR with positive and negative controls.
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