IGEM:Peking/2007/Switch: DNA digestion

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Contents

endonuclease enzymatic reaction

Buffer

There is great influence on enzymatic efficiency by enzymatic buffer. We should choose buffer according to the double digestion form provided by Takara and different activities for different endonuclease in various buffer.

enzymatic temperature

Most endonuclease has the optimal activity temperature of 37℃. There is exceptions such as SmaI's best temperature is 30℃ while SfiI needs 50℃ to work efficiently.

enzymatic system

Includs plasmid/PCR product, enzyme, buffer and water, sometimes with BSA and Trition X-100 in it. The system for enzymatic test should be no more than 20uL. The system for recruiting vectors and fragments should be around 100uL.

enzymatic DNA concentration

The vector concentration should be no more than 1ug, PCR fragment concentration should be no less than 1ug.

Star activity

Some endonuclease will have a less specific substrate selection under certain conditions. It will cut different sequences from original recognitions. This is called star activity.

In order to reduce the star activity, glycero concentration should not be too high and DNA concentration should not be too low. In the storage of enzyme there is glycerol. So the maximal amount of enzyme added in a system should be 10%, especially for enzymes with star activities.

The influence of Methylation

Some endonuclease activity will be affected by methylation of DNA.

There is a methylation form we can refer to.

PCR末端酶切

PCR末端酶切效率低,因此通常要在PCR末端的酶切位点外加保护碱基,具体参照PCR末端酶切效率表

失活条件

进行连接前需要先将酶失活,不同酶的失活条件不同,具体参见失活条件表。

酶切时间

酶切检测通常1hr已经足够。切载体和PCR片段由于需要尽可能消化干净,往往要将酶切时间延长至16hr。部分酶失活很快,需要在酶切过程中补加,具体参照长时间酶切残存活性表。

NOTICE

勿将酶拿出冷冻室,如要短时间拿出一定要用冰盒。

防止不同酶间相互污染

将酶稀释在Buffer中后,如不能马上进行反应,应将反应液保存在4℃

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