i design primer exactly with allel id software and try different annealing tempreture.i work with transvription factor in rapeseed
I've always used Primer3 with great success. The primers it gives you can be double-checked using the In-Silico PCR tab under the UCSC genome browser website. This will confirm that only one target will amplify. If you design primers to amplify over exon-exon junctions for expression analysis, the In-Silico PCR test should give zero results to indicate pcr failure on genomic DNA. I set my Tm's on Primer3 for 60C and run my reactions with that annealing temperature.
I use primique for designing real time PCR primers. Primer3 is also a good software. You can remove smears by loading lower amount of your real time pcr product
As already suggested, a good software like Primer3 to design primer is the first step to avoid dimers. Nevertheless, dimers may appear. Try different primer concentration (both, less or higher concentrated may help) to reduce additional bands or dimers. Also, higher annealing temperature increases the reaction specifity.
Smeared products can be interpreted as incomplete PCR products - maybe increase your extension time.
Best, Jan
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