Please post a picture of the gel. The problem may be that some unincorporated primer from the first round of amplification is remaining and causing the interference. Also, if these primers are against human or other genomic sequences you must ensure that there is no DNA contamination of the PCR mix. Use fresh PCR reagents, dNTPs, water, and highly pure template. A few other things to check:

1. Is the extension time long enough? If you have a longer fragment in the second round of PCR you may need to increase your extension time.

2. Make sure to use only a very minimal amount of plasmid in your initial PCR, like less than 1 ng. Plasmid remaining in the PCR reaction could be acting as template for the second round amplification.

3. You may have to adjust the Tm, generally increasing the Tm will lead to fewer nonspecific products

4. Reduce magnesium concentration, lower magnesium concentration increases the specificity of PCR

5. You may need to redesign your primers: you have 2 primer pairs which are used in the first round(s) of PCR. Make sure that primer pair 1 has no cross-hybridization to product 2 and that pair 2 has no cross-hybridization to product 1. Specificity can be increased by increasing the primer length and the Tm of the PCR reaction.

6. Try adding 1.4 M betaine to the reaction if none of the above are working.

Hi..

I agree with @zachery...!!!

Decrease Mgcl2, add Betaine.

Also use fresh 1X TBE buffer, Agarose gel and fresh reagents to prevent contamination.

Best of Luck..!!