质粒构建第一步就是设计引物。首先大体了解一下引物设计的一些通用原则：
1. 引物长度一般在15~30 bp，最常用的是18~27bp；
2. 引物GC含量在40%~60%之间，Tm值最好在72度℃左右。Tm值简单粗暴的计算方法为Tm=4×(G+C)+2×(A+T)，或在此基础上减5度；
3. 引物3’端不能选择A，最好选择T，G/C的错配率介于A/T之间；
4. 以上为最主要的原则，另外还要注意引物的特异性、引物内部不能形成二级结构等原则，具体可以百度之；
当然以上原则并不一定非要完全符合，但是作为一个新手而言初期最好是严格遵守为妙。下面介绍如何利用Primer5设计出满意的引物
1. 如下图，首先点击File->New->DNA Sequence
2. 点击空白处Ctrl+V粘贴我们的CDS序列，选择As is，即我们复制的是什么样的序列就粘贴的什么样的序列。下面的依次是“反向序列粘贴”、“互补序列粘贴”“反向互补序列粘贴”。
3. 点击左上角的Primer，如下图，S=Sense(上引)，A=Antisense（下引）
4. 如果对现在的引物不满意，还可以点击Edit Primers编辑序列，如下图，我们删除掉末尾三个碱基，之后需要先点击Analyse，然后才能点击OK，我们可以看到正向引物已经从25个碱基变为22个碱基了。
最后点击Edit->Copy->Sense Primer，粘贴到Word中即可得到正向引物，反向引物Copy之后粘贴也会自动变为5’到3’的序列。所以非常方便，Primer 5还有很多强大的功能，大家可以自行探索。
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