How do I denature Herculase II enzyme? (Alternatives welcome)
Posted 01 November 2011 - 08:31 AM
I'm currently looking into solutions, and a few ideas present themselves:
1) Denature Herculase. I've been looking for ways to do this, but so far my searches haven't turned up anything.
2) Perform a protein/DNA separation. But since I'm working with a small library that I'm doing selections on, I need to be really concerned with yield. Also, it's hybrid DNA: ds linked by the now displaced hairpin to highly modified ssDNA. Will normal separatory techniques work? I'm not sure. I can try though.
3) Switch to yet another enzyme. But what's a good one? I need an enzyme that excels at strand displacement of GC-rich regions and that can easily be heat-killed or otherwise rendered permanently inert. Is there a more appropriate enzyme I'm just not thinking of?
Thank you for your help.
Posted 01 November 2011 - 11:03 AM
Posted 02 November 2011 - 08:45 AM
Posted 02 November 2011 - 09:07 AM
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
Posted 02 November 2011 - 09:12 AM
Never worked with Herculase, but perhaps it works if you remove the Mg2+? I.e. adding sufficient EDTA.
Interesting possibility. I'll definitely look into that. Question is, will Vent (exo-) also work without Mg2+? Then again, I could add a supply of Mg2+ for the hot start rather than enzyme. I'll need to switch to preparing my own buffer rather than using the commercial one, but it may be doable. But first thing's first: I'll need to figure out if Herculase II requires Mg2+.