I had cut two PCR products, very similar to each other, whit the same both enzymes (EcoRI and SalI), ate the same time. One of them dissapeared...twice!!! While the other is already cloned in a plasmid...can anyone help me whit this mistery? Thank you a lot!
Hi there,
One of your samples is contaminated with DNAse which fully degrades the product during incubation.
If the pcr product is small and there are internal cut sites then you may have cut into many small fragments.Each small fragment will take up less EtBr and if the original amount of uncut pcr product was only weakly visible then the smaller products will not be visible at all.. More details are needed about the size of the fragment, number of cut sites and the amount that you are cutting.
Thank you, people!
The PCR product is aprox 800pb long, and has no internal sites for the RE used (I double checked). About DNAse, it could be, but I made exactly the same cut at the same time whit another alele of this genes (very similar), used the same reactives and worked just fine...so I guess that´s not the problem.
I didn´t try individual and consecutive cuts, so I´ll try that option ...I let you know!! Thanks again!
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