TheprotocolforLICbyExonucleaseIII

The protocol for LIC by Exonuclease III
梁耀极
1. Design the primers with 15-bp overlap;
2. Digest the vector by proper restriction enzyme;
For getting high quality vectors, there are some good advices as follows:
1) The restriction sites we choose should be 5’-overhangs or
blunt ends , but it shouldn’t be 3'-protruding ends ;
2) Digestion by double enzymes;
3) Digestion the vector overnight to make sure complete cleavage as possible;
4. Quantitate the concentration of the vector through running DNA gel and the vector is
prepared for LIC;
5. Amplify the insert by PCR with the overlap primers, gel-purified, and quantitation; the
insert is also prepared for LIC;（You can also treat the templates with DpnI, if there are
scarcely any background bands and the positive PCR bands are dense and special
enough.）
6. Mix the vector and insert in the reaction system as follows:

Vector 50-100ng
Insert 50-100ng
10ⅹExoⅢ buffer 1ul
Add ddw to 10ul

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 several times ;
9. Place the tube on the ice bath or 4℃ for 60mins;
10. Add 1ul 0.5M EDTA (pH 8.0) to stop the reaction; pipe the mixture for several times;
11. The mixture is then melted at 65℃ for 5mins;
12. Place on the ice bath for 5mins;
13. Centrifuge to concentrate the mixture;
14. Transform the mixture of DNA into DH5α;
15. Incubate the bacteria on the LB plates with proper antibiosis for 16h;
16. Pick up single clone for mini-preparation, analyze by restriction enzyme and further analyze by DNA sequencing.