I am using a drosophila genome extracting a particular gene. I tried using fresh DNA, sterilized water and my negative control is perfectly alright. Each time i repeat my pcr the results varies; single and double alternately. Quantity of PCR mixture is the same for each reaction. What would be the possible problem?
This is just a simple suggestion. You could increase the annealing temperature so make your PCR more specific. You may lose sensitivity but gaining specificity. And dilute your template DNA may help too.
Genomic DNA?
Is your DNA from the same extraction, or is it a new extraction each time?
Could this be an allelic difference (for example with a difference in intron lengths)? Or maybe a pseudogene?
Check if your primers are on two different exons. If so, you should consider purifying and clone/sequence the PCR product.
- Badges
- Science method