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| I would guess that the false positive could be due to one of two reasons. You could blast your primers against the Arabidopsis genome to see if, by chance, there is 500 bp region flanked by sequences similar to one or both of your primers. You could also check the sequence or restriction pattern to confirm that the 500 bp fragment is what you expect. More likely, one or more of your pipettors is contaminated (from pipetting the plasmid or PCR products). Did you do a no-DNA control? If you also see the product, then you'll probably have to replace your reagents and clean your pipettors (especially inside the shaft) or borrow someone else's for setting up the reactions. (UV treatment has been shown to reduce contamination, too.) Pipettor contamination can happen surprisingly easily, especially if you pipet up and down to mix in dye or to rinse tips between loading samples on the gel. I routinely use a different pipettor for loading PCR products on gels. Good Luck, Mike |
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| arabidopsis , hygromycin |
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