I am trying to make dilutions from a commercially available plasmid stock of plasmid pBR 322. The buffer is Tris and EDTA pH 8. After I prepare the buffer, should I autoclave it for it to be DNAse free?
What type of water is good for standard buffer preparation for molecular biology research? I am Organic Chemist and beginning to work in the field of plasmids, so some guidance will be highly appreciated.
Thanks and kind regards
Ankita
Hello Ankita Ray,
1 mM EDTA in your Tris buffer should inactivate most of DNAses in solution. After dilution of plasmid DNA make some aliquots and store at -20°C. For buffer preparation I use Millipore ddH2O. Autoclaving is good way to 100% inactivate DNAses, but if you do not have access to autoclave I think it is not a critical point assuming you work in gloves and use pure noncontaminated Tris :-)
Good luck!
Thank you Martin,
Would you then suggest autoclaving water prior to the preparation of HEPES buffer? Although my dilutions will be made in tris buffer but for some other applications I would then require to use HEPES buffer. And yes, we do have access to autoclaves, I just wasn't sure to what extent it was quintessential.
Thanks for your kind help.
Ankita Ray,
see this link: https://chemistry.stackexchange.com/questions/15866/what-happens-if-you-autoclave-hepes
I would recommend to autoclave water prior preparation of HEPES as you suggest.
Best regards
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