一、In a microcentrifuge tube prepare a solution of linear DNA (25-50ng) in deionized water or TE buffer (10-35µl). 二、Add: (一)10X ligation buffer 5µl, (二)50% PEG 4000 solution (for blunt ends only) 5µl, (三)deionized water to 50µl, (四)T4 5u. Vortex the tube and spin down in a microcentrifuge for 3-5 seconds. DNA Ligase 三、Incubate the mixture for 1 hour at 22°C . 四、Inactivate T4 DNA Ligase by heating reaction mixture at 65°C for 10 minutes. 五、Resulting reaction mixture can be used directly for transformation. References Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 1994-2001. |
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