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 ;
2) Digestion by double enzymes;天蝎座和射手座
3) Digestion the vector overnight to make sure complete cleavage as possible;
桂花泡水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:
Vector 25-50ng
Inrt 25-50ng
江汉路Add ddw to 10ul 10ⅹExo Ⅲ
buffer 1ul
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);
交相辉映的意思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;
睡觉前运动好不好
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;平方米平方公里
12. Place on the ice bath for 5mins;
13. Centrifuge to concentrate the mixture;
扫墓祭祖14. Transform the mixture of DNA into DH5α;
emptied
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.
