- DNA连接酶的最佳工作温度是25度左右,也就是室温。而把DNA两端连接在一起的最佳温度(optimal temperature)是1度左右。这样就出现了一个很大的温度差。到底选什么温度呢?根据个人经验,大家可以先在室温下孵育一个小时,然后把混合物放在4度冰箱里一晚上。这样效率大大提高了。当然,大家还要考虑连接反应中DNA片段是带有互补粘性末端的还是平末端的。
- 连接缓冲液(ligation buffer)里含有ATP,也是整个反应中必不可少的。但是ATP会随着时间的增长而降解,尤其是在反复冻融循环时(freeze-thaw cycle)。所以大家做这个实验的时候千万不要偷懒,一定要提前把连接缓冲液一次性融化之后分成小份储存(make aliquots)。在使用前,将一份分装的缓冲液放在室温融化或用手掌温度辅助融化,然后放在冰里。但是千万不要加热融化,否则ATP还是会降解,分装的精力也白费了。
- 不但要有对照(controls),而且还要尝试不同的 vector: insert 比例。因为每一个连接反应在分子水平发生时都是不完全一样的,照搬网上或是别人的实验方法肯定是行不通的。大家不妨在做连接反应的时候,尝试着使用下面表格中提到的条件,然后把含有质粒的连接反应物在大肠杆菌中进行转化(bacterial transformation),通过菌落的多少来检验连接效率。
Vector | Insert | Insert: Vector | DNA Ligase | 注释 | |
实验1 | √ | √ | 1:1 | √ | 这是常用的比例。大家需要自己通过实验来找到最优比例。 |
实验2 | √ | √ | 3:1 | √ | |
实验3 | √ | √ | 10:1 | √ | |
控制 1 | √ | – | – | √ | 对没有被酶切或是重新连接在一起的质粒进行定量。 |
控制2 | √ | – | – | – | 对没有被酶切的质粒进行定量。 |
以上技巧是小编根据个人经验和网上资料整理得来的,如果有更好的建议和意见,也欢迎各位小伙伴留言告诉我们。
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