I did tail biopsy to genotype a particular gene. The result came out positive for my desired gene but two days after using same lysed genomic DNA, it came out negative. In my confused state, l decided to repeat tail biopsy, Genomic DNA lysis and PCR today, the result came out Positive for the desired gene all under same PCR conditions, primers and Taq polymerase. What could be the cause or causes of this variation?
I agree with much of the above but would not store my DNA in water, Water absorbs CO2 from the air and becomes acidic causing depurination and degradation of the dna, Storage in TE to remove magnesium ( needed for nucleases to work ) and buffered alkaline at pH 8,4 is a good storage medium
Hello Joshua,
the first thing that comes to my mind are nucleases in your final elution buffer. If you use nuclease-free TE than you can store your DNA at 4-8°C, if you usie nuclease-dree water that you should store your DNA at -20°C without repeatedly thawing-and-freezing (at least that´s what they all say). I always keep my DNA in water at 4°C. It happens to me, too, sometimes :)
Best wishes
Dirk
If you are not already, you should be including no-template controls with each PCR experiment. Your reagents or workspace could be contaminated with free template DNA that would result in a false positive. If you get no amplification in your no-template control, you can be confident that your experiment is not giving you false positives.
A false positive is due to contamination. Throw away ALL of your reagents, clean your work area and label everything carefully. Always include a set of positive and negative controls in your PCR to help interpret your results.
A false negative is a failed reaction. It means a key step or component is missing or degraded. Most likely is degraded DNA, you used the wrong primers or forgot to add a reagent.
Store your reagents carefully, keep a clean work space, aliquot reagents to avoid freezing and thawing and take good notes so you can trouble-shoot.
Prof Dirk Schmidt Govinda Sharma and Katie A Clark, am grateful for you answers to my question. They are really helpful.
I agree with much of the above but would not store my DNA in water, Water absorbs CO2 from the air and becomes acidic causing depurination and degradation of the dna, Storage in TE to remove magnesium ( needed for nucleases to work ) and buffered alkaline at pH 8,4 is a good storage medium
Actually it happens to me as well, some negative samples be positive after a while when I run electrophoresis again!
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