多丽丝莱辛The protocol for LIC by Exonuclea III
1. Design the primers with 15-bp overlap;
2. Digestion the vector by proper restriction enzyme;
For getting high quality vectors, there are some good tips as follows:
1) The restriction sites we choo should be 5’-overhangs or
blunt ends , but it shouldn ’t be 3'-protruding ends ;
汤圆煮多久才熟
2) Digestion by double enzymes;
溥仪皇帝3) Digestion the vector overnight to make sure complete cleavage as possible;
4. Quantitation the concentration of the vector through running DNA gel and the vector is prepared for LIC;
5. Amplifying the inrt by PCR with the overlap primers, gel-purified, and quantitated, the inrt is also prepared for LIC;（You can also treat the templates with DpnI, if there are scarcely any background ba
nds and the positive PCR bands are den and special enough.）
6. Mix the vector and inrt in the reaction system as follows:
滂沱的读音Vector 25-50ng
Inrt 25-50ng
Add ddw to 10ul 10ⅹExo Ⅲ
buffer 1ul龙虎山旅游攻略
7. Place the tube in the ice bath for 5mins
( Make sure the mixture in the tube is cooled to the temperature of the ice bath, and the following approach should also operate on the ice);
8. Add 1ul ExoIII(20units) to the reaction mixture; pipe the mixture for veral times ;
9. Place the tube on the ice bath or 4℃ for 60mins;恨别
10. Add 1ul 0.5M EDTA (pH 8.0) to stop the reaction; pipe the mixture for veral times;
11. The mixture is then melted at 60℃ for 5mins;
12. Place on the ice bath for 5mins;
工程联系单13. Centrifuge to concentrate the mixture;
14. Transform the mixture of DNA into DH5α;
爱活着15. Incubate the bacteria on the LB plates with proper antibiosis for 16h;
16. Pick up single clone for miniprepare ,analyze by restriction enzyme and further analyze by DNA quencing.
