Site directed Mutagenesis-Primer Design

We received proteorhodopsin sequence from Kwang-Hwan "Kevin" Jung, Ph.D., Associate Professor, Department of Life Science and Institute of Biological Interfaces, Sogang University with three restriction sites EcoRI, SpeI and PstI. Hence the plasmid had to be mutated for iGEM compatibility. Hence we had to design primers for site directed mutagenesis at each of these sites. So we have come up with a Primer design tool for SDM as a Criteria:

Mutations in the middle of primer

Tm difference should be less than 5

No 3' Breathing should be ensured by keeping G or C at the end