What is your solution regarding extra bands in ITS molecular markers?
I am trying to use ITS1 and ITS4 markers in 15 plant species. Unfortunately, I only received extra bands instead of one after examining my PCR products. In such kind of positions, sequences will be with lots of noises.
I decrease Forward and Reverse primers as well as DNA ingredients within the reaction. Again two bands were observed.
In the second stage, the initial annealing temperature was 58, I increase it to 60, and recently I increased it again to 61. In this case one of my plant samples showed only one band, but the second did not multiplied successfully.
What is the best and the easiest solution to this problem? I want to gain only one sharp band, instead of two.
Best Regards
Hello,
One of the first solutions you found is to increase the hybridization temperature, which makes it possible to eliminate some of the parasitic hybridizations of the primers.
However, according to the second result, you have a sequence, in another location on your DNA, which has similarities with your primers, and in this case, the simplest is to directly recover your target band, by cutting it directly on gel and purifying it afterwards.
Hello,
Have you ever tried gradient pcr by calculating annealing temperature and if you not tried you should try one time with minimal concentration of plasmid DNA
Vishnu Dutt
So many thanks for your answer. How do you calculate annealing temperature? Do you have any protocol?
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