If possible redesign the primer that has a 9 base homology to the wrong place. If not then using these primers use a hot start enzyme so that the poor homology is less likely to anneal and run a gradient of DMSO at 1%,2%,3% up to 8% and hopefully at one of the dmso concentrations you will have only the clean band..

prepare your reagent mixes on ice and load the tubes into a pcr machine already at 80c to minimise low temperature primer annealing and try an assay with 1/4 the amount of primer again to minimise unwanted primer annealing.

If all else fails try minimising the low temperature steps of the pcr to favour the short amplification so anneal for 10seconds and extend for 10 seconds.

You can also try shortening your annealing and extension times (15 seconds each). Smaller products are typically made faster than larger products & you might be able to create fewer copies of the unwanted size amplicon.

Or just design new primers.

Hi there,

if the purpose is to get the 120bp amplicon then just don't bother: gel purification will perfectly do as it is efficiently produced!

I eliminated the NSP by hot starting at 95c. As seen in my 45s lane (same conditions as original gel from my question but hot started). Dropping the anneal and extension times still made consistent product and should work to save me some cycle time (see lanes 30 20 and 15 seconds) Thank you Katie A Burnette & Paul Rutland

Lovely result. Well done Ryan Scanlon

Ryan Scanlon - Looks like you got it! Good progress. Another thought in case the bands reappear, if the product you are trying for is short you can also reduce the extension time to reduce the likelihood of read-through. For most polymerases the company will list "xx" seconds of extension time per kb. If the company list 15 sec/kb extension time, it is just an average. Some templates will need more and some will need less. However, if it lists 15 sec/kb for the enzyme and your expected product is only 200 bp but you are using an extension time of 45 sec, then there is a huge risk of getting longer bands that are not a result of mispriming. They will likely only go away permanently if you reduce the extension time to appropriately match your product length.