I am designing primers to clone membrane protein homologs from several archaea organisms in E. coli. The templates sequences are very CG enriched, resulting in high Tm temperatures, %CG and dimers. DO you have any tips for improve primer design for archaea?
Hi, Yasser. I am working with archaea tRNA cloning. The Tm of my primers are 70-72 degree. Normally they are fine with standard PCR using KOD hot start DNA polymerase. If possible, you could design a pair of primers and try PCR first. If it does not work, you might have to spend some time on troubleshooting. People add DMSO into PCR to help denature high %GC template.
Here is the guidelines for PCR by NEB. Hope this helps!
template. https://www.neb.com/protocols/2012/06/01/guidelines-for-pcr-optimization-with-phusion-high-fidelity-dna-polymerase
- Badges
- Science method