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Digestion of PCR and RT-PCR Products With Restriction
Endonucleases Without Prior Purification or Precipitation
By Gavin R. Turbett* and Loryn N. Sellner
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.
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