My DNA(plant) was extracted using CTAB buffer and the DNA was dissolved in TE buffer. DNA was stored at -20°C and it is going to be 2 years now from the date of extraction. All were going smoothly PCR, everything. But today when I mixed my template to my PCR mixture, which I usually do at the last step, turned light orange color. I don't understand how this is happening. And even with loading dye the same is happening. Is there any wrong with the template or with the TE buffer. Please help!!
What polymerase are you using? I remember Thermo fisher introduced a polymerase that changes color upon template addition.
Not sure how to explain the loading dye!
you often find this when running old running buffer again and running gels too far and too fast because the buffer becomes acidic at the cathode and these dyes are pH indicators and turn orange in acid solution. If it turns orange when added to your sample then you have too much acid in your samples so check where an acid pH could arise in your samples and also check that your running dye is dissolved in TE/TAE/TBE buffer and not in water as the buffer will neutralise excess acid in the samples
I am using GeneTaq green DNA polymerase from PUREGENE (Genetix Brand). And such a thing never happened before.(Mahmudul Hasan)
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