G.tigrina Hox gene DthoxC insertion into prokaryote E.coli / (by UNIamCloning)/2011/11/04

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Primer Design

  • Today our primers were designed to allow for the removal of the stop codons in the GFP biobrick part as well as join the two biobrick parts together (GFP in front attached to ATF1). It was decided to use Vf and Vr as the forward primer for the GFP part and the reverse primer for the ATF1 part since both these sights are already found in their vectors (this will also save us the trouble of designing our own forward and reverse primers including the prefix and suffix parts). Next, we designed the reverse primer for the GFP part (E0040) including the enzyme KPN1 plus an additional two base pairs for additional overlap: 15_E0040_R 5'-atccatggtttgtatagttcatccatgcc-3'. Finally, we designed the forward primer for the ATF1 part also including the KPN1 enzyme plus two additional base pairs: 15_J45014_F 5'-atggtaccatgaatgaaatcgatgagaaaaat-3'. After running both these primer sequences through the IDT website it was noted that hairpin formation was minimal/unlikely and the melting temperatures of both were very similar.