1st denatureation: 94C for 3 minutes by annealing: Most primers will glow best on 55C in 30 seconds even if the ideal glow temperature is priming TM minus 5c. Find the order of the desired restriction enzymes available on the manufacturers sites for Forward drivers figure. Inhibiting contaminants from PCR product or linear vector both PCR product and linezed vector should be cleaned. See the article PubMed PubMed Central Google Scholar Yamaji N Huang CF Nagao S Yano M Sato Y Nagamura y MA JF. Euphytica. 2000 113:1635. See the article Google Scholar Matos M Prez-Flores V Camacho MV Pernaute B Pinto-Carnide O Benito C. Initial denaturation: 94C for 3 minutes Annealing: Most primers will anneal best at 55C for 30 seconds although the ideal annealing temperature is the primer Tm minus 5C.Find the sequence of the desired restriction enzymes available on the manufacturers websites for the forward primer Figure.Inhibitory contaminants from PCR product or linearized vector Both the PCR product and the linearized vector should be purified.View Article PubMed PubMed Central Google Scholar Yamaji N Huang CF Nagao S Yano M Sato Y Nagamura Y Ma JF.Euphytica. 2000 113:1635. View Article Google Scholar Matos M Prez-Flores V Camacho MV Pernaute B Pinto-Carnide O Benito C.