Difference between revisions of "IGEM:Peking/2007/Switch: DNA ligation"

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(New page: ==连接== 1.普通连接的体系为10uL,其中片断+载体5uL,Ligation Kit 5uL。 2.Ligation Kit需在冰盒中溶化。分装至1.5mL离心管中使用。 3.先加片断...)
 
(连接)
 
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==连接==
+
==DNA ligation system==
  
1.普通连接的体系为10uL,其中片断+载体5uL,Ligation Kit 5uL。
+
1.10uL, fragment+ vector 5uL, Ligation Kit 5uL.
  
2.Ligation Kit需在冰盒中溶化。分装至1.5mL离心管中使用。
+
2.Ligation Kit should be separately loaded into 1.5mL EP tubes for any usage.
  
3.先加片断,后加载体。
+
3.First add the fragment, then add the vector.
  
4.管口用Parafilm封好。
+
4.cover the tube cap using parafilm.
  
5.16℃反应1hr以上。
+
5.16℃ reaction more than 1 hour.
 
 
注意:连接酶25℃达到最大活性,3min失活。4℃活性较低,但可以保持过夜。16℃的性质在二者之间。4℃连接过夜的效果最好。根据片断与载体连接的难易程度选择连接的温度与时间。
 

Latest revision as of 04:17, 20 October 2007

DNA ligation system

1.10uL, fragment+ vector 5uL, Ligation Kit 5uL.

2.Ligation Kit should be separately loaded into 1.5mL EP tubes for any usage.

3.First add the fragment, then add the vector.

4.cover the tube cap using parafilm.

5.16℃ reaction more than 1 hour.