Is there a reason why you need to do nested PCR since that will contribute to background? Also 35 cycles is quite a lot, you might try reducing to 30 or even 25.

Hi

From your gel A it is possible to see that the neg ctrl doesn't show the smear, so it means no carry-over contamination which is brilliant.

You just need to optimize you PCR conditions because this smear might be due to many things (too much starting material, too much Taq, too many cycles, Mg2+ final concentration). Were your primers blasted to make sure they are specific?

The other thing is your "sample I" in gel A doesn't have the smear, is this sample less concentrated than the others, if yes you might just need to dilute more your other samples.

As for non-specific binding of your primers...test the appropriate melting temperature by doing a gradient and seeing which one is optimal.

If no difference, try to reduce the Mg2+ concentration.

Hope it helps.

Diana

Many possibilities are there

As i can see from your gels there is nothing on your -ve controls, thre is no contamination in your PCR reagents.

your template is a former PCR product - right

In your case it seems like too much template in your reaction- so set up two reaction tubes

1) 0.5 micro lit of PCR product as template

2) 1 micro lit of PCR product

and the annealing temp between 58 to 60 or 55 deg C(Should work on almost in all reactions)

Try this this should definitely work for you otherwise let me know we can find other solution

Good luck

Cheers

Aditya

Thank you Diana , hope it works , i will try again with less amount of Dna, i guess i have taken too much of dna, as my samples is of High concentration Dna.

You have asked a very concrete, detailed question I wish to see always on RG! Also your picture is nicely showing, probably to much of template was taken as mentioned above. If it is my experiment I would play with DNA concentration only. Keep fingers cross - Jan

Is there a reason why you need to do nested PCR since that will contribute to background? Also 35 cycles is quite a lot, you might try reducing to 30 or even 25.

First question: Why do you use nested PCR? Is this really necessary?

What then comes to my mind are two things: You are using a lot (virtually a ton) of DNA as a template. Try using 0,5-1ul of you first PCR for the nested, this should still be enough DNA to amplify. Is your final product 1023 bases long? If so, why do you reduce the amplification time in the nested PCR step to 45 sec? Standard taq polymerase (which I assume you use) needs around 1min per kilobase of DNA, so this is simply to short. You can see two bands on some of your gel lanes, one around 1kb and the second around 700-800.

Thanks a lot everyone ,...finally I got the desired bands , problem was I was taking too much dna .