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         <br/><p><b>PCR</b></p><br/><p>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 1: Design of appropriate forward and reverse primers<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 2: Prepare our template<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 3: Prepare the PCR mix. (Kapa Hifi PCR kit.)<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 4: Run PCR<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 5: Examine the results by electrophoresis<br/>

         Note: If the template is genomic DNA, we would adjust the annealing temperature at 45°C. It is because the copy number of target gene may be low. We use this annealing temp when perform PCR of Tir1, 26s, 5.8s ITS</p>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 1: We design primers for parts with prefix and suffix.<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 2: Perform PCR and cleanup the PCR product<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 3: Before insert our parts into standard backbone, pSB1C3, we perform RE digestion to make sticky ends of both inserts and backbones.<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 4: Ligation of inserts and backbones<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 5: Transform our ligation products into DH5α and streak the transformed DH5α on LB agar plate with chloramphenicol.<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 6: Inoculate single colony into broth with chloramphenicol.<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 7: Miniprep the plasmid DNA from the overnight broth culture.<br/>

        &nbsp&nbsp&nbsp&nbsp&nbspStep 8: Confirm the products by both RE digestion and PCR sequencing<br/></p>