A possible false positive reaction can occur if test plan sample are contaminated or infected. As PCR is a kind of enzyme reaction, any factors that affect an enzyme reaction could also result in a false PCR. These factors include improper preparation of DNA template, primer and reagents, and existence of interference. A false negative could result from the above factors.
- Extra bands can be contamination but are also at least as likely to sub optimal PCR conditions
- In particular a mismatch between your primer Tms and annealing temp:
- In general you want your annealing temp to be some where between 2C and 5C lower than your actual primer melting temp (Tm) for specific but efficient PCR; Check that your annealing reactions are not > Tm -5C as this could result in non specific bands
- Also, make sure you have subjected your primers to BLAST searching to verify they only hit one target (if over 20bp invariably they do)
- As you are obtaining extra bands for what ever reason
To add to the above excellent answer, you should also include a full set of controls (positives and negatives) every time. Your data are only as reliable as your controls.
I agree with the answers above and really think that adjusting the PCR conditions may help a lot in overcoming the issue. Usually, one Celsius degree of temperature may make difference in your results.
I agree with the previous answers. In case try a touchdown PCR after you found the proper Tm.
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