Hello. For the lysis step, some single worm PCR protocols are using 60 °C and some are using 65 °C. Any ideas why? Some non-C. elegans protocols use 65 °C for heat inactivation of proteinase K. The web suggests a range of temperature for optimal activity from 37 °C to 60 °C. Anybody want to clarify this for me? What is the benefit of 65 °C? Thanks.
I have been using protinease K (from invitrogen) at 60 degrees for an hour for years and have had excellent results. Likely you could test different temps, but if it ain’t broke don’t fix it.
There’s different types of proteinase K which have different optimal temperatures, some have been engineered to have better stability than others.