id 24 for isoform sst5c the four pcr reactions were carried out concurrently using a program consisting of an initial 2 minute denaturation at 94 u00b0 c followed by thirty four 30 second denaturation cycles at 94 u00b0 c 30 seconds of alignment at 62 u00b0 c and 40 seconds of extension at 72 u00b0 c in every case a high fidelity polymerase was used with a pfu ultra stratagene type copy supplementing the reactions with 1m betaine sigma through these pcr reactions a sequence of restriction for the hindiii enzyme was able to be incorporated into the 5 u2032 end of each e1 the 3 u2032 blunt end of the e1 the 5 u2032 blunt end of the e2 and a sequence of restriction for the bamhi enzyme in the 3 u2032 end of each e2 it was previously verified that none of the enzymes of restriction had additional cut sites within the sequences of interest the pcr fragments obtained were purified by using the quiaquick mini elute quiagen commercial kit and after enzymatic digestion with hindiii and bamhi they were linked through a triple reaction within the pcdna3 invitrogen eucaryote expression vector which had been linearized with the previous enzymes of restriction both sst5b pcdna3 and sst5c pcdna3 structures were sequenced at least in duplicate in order to verify the integrity of the sequences and to compare them with the gi13937340 genomic sequence which.