One-Tube PCR Cloning Method

Chun-Ming Liu

This method has 3 reactions (restriction, polishing and ligation) carried out in one tube using ligation buffer. Believe or not, it works very well for me and a lot of my friends.

Purify PCR product using Spin-Column S-300 from Pharmacia.
Ligation mixture (you may be able to reduce the cost by reducing the volume):

40ul purified PCR product
2ug uncut pBluescript SK(+)

20 units EcoRV
20 units T4 DNA polymerase
25 units T4 Ligase
2ul 2mM dNTP
14ul 5X ligation buffer
add water to 70ul

14 - 17^oC for one to two days (2 days are better).

Transform directly to DH5a ultra-competent cells using my favorite 5 minutes method.

Comments or Questions? Send to me at:chunming.liu@wur.nl