IGEM:Peking/2007/Switch: DNA digestion

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(New page: ==限制性内切酶反应== ===Buffer的选择=== 酶切效率受Buffer影响很大,Buffer的选择参照各种限制性内切酶在不同Buffer中的活性及双酶切Buffer表 ===酶...)
Current revision (07:16, 22 October 2007) (view source)
(enzymatic DNA concentration)
 
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==限制性内切酶反应==
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==endonuclease enzymatic reaction==
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===Buffer的选择===
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===Buffer===
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酶切效率受Buffer影响很大,Buffer的选择参照各种限制性内切酶在不同Buffer中的活性及双酶切Buffer表
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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.
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===酶切温度===
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===Digestion temperature===
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多数限制性内切酶的最佳活性温度是37℃,但也有例外,如SmaI的酶切温度为30℃,SfiI的酶切温度为50℃。
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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.
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===酶切体系===
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===enzymatic system===
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通常的酶切体系包括质粒/PCR产物,酶,Buffer和水,有时也包含BSA和Triton X-100。酶切检测体系不大于20uL,切载体和PCR片段的体系通常为100uL。
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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.
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===酶切DNA浓度===
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===DNA concentration in digestion system===
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检测用质粒浓度以酶切后最小片段可见为宜。
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The vector concentration should be no more than 1ug, PCR fragment concentration should be no less than 1ug.
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载体浓度不大于1ug,PCR片段浓度不小于1ug。
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===Star活性===
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===Star activity===
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限制酶在一些特定条件下使用时,对于底物DNA的特异性可能降低。即可以把与原来识别的特定的DNA序列不同的碱基序列切断,这个现象叫Star活性。
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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.
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尽量减少高甘油,低DNA浓度的情况有助于限制Star活性。由于酶保存液为甘油,加酶体积最大为体系的10%,对于有Star活性的酶应当更低,且适当增大DNA量,减少酶切时间。
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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.
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===甲基化影响===
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===The influence of Methylation===
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部分限制性内切酶活性受甲基化影响,具体参照甲基化影响表
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Some endonuclease activity will be affected by methylation of DNA.
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===PCR末端酶切===
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There is a methylation form we can refer to.
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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
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===失活条件===
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===Terminal digestion of PCR product===
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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。
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The terminal digestion always has a low efficiency. Some additional base pairs are always added at the end of PCR product during primer design in order to improve the digestion efficiency.
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===酶切时间===
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There is a form about PCR terminal digestion.
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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。
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===Inactivation===
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After the digestion, endonuclease must be inactivated before applied to ligation system. Different enzymes have different inactivation condition.
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There is a form about inactivation conditions of all usual endonuclease.
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===Digestoin Timing===
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1 hour enables a test digestion. If digestion is used for cutting vectors and PCR fragments, in order to digest thoroughly, about 16 hours is a relatively good choice. However, some endonuclease used in the digestion system inactivates quickly. So we need add new enzymes at intervals during digestion.
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There is a special form of different endonuclease about their remaining activity after different periods of time.
===NOTICE===
===NOTICE===
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勿将酶拿出冷冻室,如要短时间拿出一定要用冰盒。
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DO NOT carry the endonuclease out of fridge. If really need to do so, please use an ice box to protect the enzymes from inactivation due to high temperature.
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防止不同酶间相互污染
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Protection of cross-pollution between different enzymes
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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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If endonuclease have already been added into buffer solution while somehow the digestion reaction is not ready to start right now, DO store the mixed solution under 4℃

Current revision

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.

Digestion 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.

DNA concentration in digestion system

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.

Terminal digestion of PCR product

The terminal digestion always has a low efficiency. Some additional base pairs are always added at the end of PCR product during primer design in order to improve the digestion efficiency.

There is a form about PCR terminal digestion.

Inactivation

After the digestion, endonuclease must be inactivated before applied to ligation system. Different enzymes have different inactivation condition.

There is a form about inactivation conditions of all usual endonuclease.

Digestoin Timing

1 hour enables a test digestion. If digestion is used for cutting vectors and PCR fragments, in order to digest thoroughly, about 16 hours is a relatively good choice. However, some endonuclease used in the digestion system inactivates quickly. So we need add new enzymes at intervals during digestion.

There is a special form of different endonuclease about their remaining activity after different periods of time.

NOTICE

DO NOT carry the endonuclease out of fridge. If really need to do so, please use an ice box to protect the enzymes from inactivation due to high temperature.

Protection of cross-pollution between different enzymes

If endonuclease have already been added into buffer solution while somehow the digestion reaction is not ready to start right now, DO store the mixed solution under 4℃

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