I assume that when you say there are bands in both positive and negative that you mean the bands are the same size in the negative as in your samples.

You have post pcr contamination in either your pipettes,reagents or working area. Buy new primers and make stocks with someone elses pipettes working in their area in case the primers have become contaminated.

Then using other researchers (or new) pcr reagents (ntp,Mg,enzyme) and also their pipettes set up a pcr with 4 no template controls and 2 positive samples. During this time clean your working area and change all of your plasticware and clean your pipettes inside and out. If the NTC samples are clean and there is a band of the right size in the positive samples then you can set up the next pcr in your area using your reagents to isolate where the contamination is or just continue using the new reagents

Are the bands in your negative control the size you expected for your positive control? If yes, then your issue is contamination. Follow Paul's advice.

Or are you just seeing unused primers? Those are typically less than 100 bp in size. In that case you need to get a full set of controls since that means your positive controls failed.

Paul Rutland

Thank you very much.

I think the contamination should be the problem.

I think so Sherry Liao . One further idea is that if all attempts to get rid of the contamination fail then a sure way of dealing with contamination is to design one primer outside ( larger pcr product) of your amplimer. Then this primer cannot bind to the old amplimer and you will get clean results again