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do primers and dNTPs alter digestion efficiency? - (Jan/02/2006 )


i have a very short question...

i want to directly digest my phenol extracted PCR product with RsaI.
do free dNTPs and primers alter the digestion efficiency?
any experiences?




Not really, in my experience. Never really noticed a difference. It might change ligation efficiency, however.



for these points, you can refer at neb web sites in "cleavage in a primer extension mix"... (here)

here is for Rsa I : + + + (means 100%)


thanks a lot for your replies.... there are restriction enyzmes that show very decreased efficiency in the presence
of oligos and dNTPs... luckily i have chosen an enyzme that doesn't seem to care...



no, it will digest your insert because you will use comptable buffer but in ligation process it will affect due to free dntps affet the activity of ligase

-sudhir singh-