Digestion of PCR and RT-PCR Products With Restriction Endonucleases Without Prior...
Digestion of PCR and RT-PCR Products With Restriction Endonucleases Without Prior Purification or Precipitation
- Comments & Ratings
We have tested 33 commonly used Promega restriction endonucleases (REs) for their ability to digest DNA directly in polymerase chain reaction (PCR) amplification buffers. In total, 29 (88%) of the REs showed complete digestion of the target DNA after overnight incubation in PCR buffer without the requirement for the addition of any other component. The remaining 4 (12%) REs required additional magnesium or the addition of restriction endonuclease digestion buffer to function adequately. The composition of the PCR buffers tested did not affect the results. The REs also functioned equally well in an RT-PCR Buffer. The results show that digestion of PCR and RT-PCR products may often be performed directly in the PCR tube without the requirement for any precipitation or purification steps. However, we do not recommend the use of this procedure for cloning applications.
Promega Notes 60, 23.
Gavin R. Turbett and Loryn N. Sellner
Molecular Biology Laboratory, Department of Pathology, Royal Perth Hospital, Perth, Western Australia
Publication Date: 1996