cboyd at holyrood.ed.ac.uk
Mon Jul 17 11:00:23 EST 2000
Martin Chan <s987517 at mailserv.cuhk.edu.hk> wrote:
: Dear netters,
: I am going to directly use the PCR mixture for restriction digestion,
: but I fear the remaining Taq in the PCR mixture will fill in the 5' overhang
: generated by restriction endonucleases. Is my worry necessary? Can Taq be
: inactivated by heating at 94-96C for 30 min prior to restriction digestion?
: I am frustrated by the low recovery of my lab's PCR purification kit, so I
: plan to skip the purification step.
No-one else has mentioned this, so I will just add that you can get rid
of Taq using proteinase K digestion. See:
Crowe, J. S., Cooper, H. J., Smith, M. A., Sims, M. J., Parker, D.
and Gewert, D. (1991) `Improved cloning efficiency of polymerase chain
reaction (PCR) products after proteinase K digestion.' Nucleic Acids
Res., 19, 184.
Chris Boyd | from, but \ Medical Genetics Section
Chris.Boyd at ed.ac.uk | not for, / MMC, Edinburgh Uni.,
http://www.ed.ac.uk/~cboyd EH4 2XU, SCOTLAND
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