For context, I am doing multiple restriction digestions (>50) and I want to streamline it by making a 10x cutsmart buffer + restriction enzyme mastermix.
Reaction volume will be 50ul so the recipe will be:
44ul - DNA + Water
5ul - 10x Cutsmart buffer
1ul - Restriction enzyme (in this case ApaI)
Essentially I want to make a mastermix of 5 ul cutsmart buffer + 1ul restriction enzyme and just distribute it to my tubes followed by adding my DNA.
Usually I would add the cutsmart buffer first, followed by DNA then finally the restriction enzyme, but it gets rather tedious when there are many digestions to be done.
My main concern is does the concentrated cutsmart buffer affect the enzyme (like inactivate it or affect it in any negative way) given that its 10x.
Thanks for your response and experience in advance.
That is fine. I just would not freeze it since the enzyme will lose its activity since you will dilute the glycerol in the enzyme buffer so just prepare fresh the amount you need. Its is also easier just to use a fixed volume of DNA so you can also add water to mastermix.
Hi there,
I personally always prepare the master mix with everything but the DNA (ie. 10xbuffer+water+enzyme) in order to have the enzyme in a diluted buffer. In order to limit the possible negative effect of buffer concentration on enzyme activity. The only constraint being I have to have the same volume of DNA in each digest.
Hanna Alalam So do you mean it should be fine to just add the enzyme directly into the undiluted buffer, so long as I do not freeze it again as the glycerol concentration will be reduced?
Dominique Liger Hmm, I could resuspend the DNA in a lower volume of water and add that to the mastermix you mentioned, the only draw back is that the amount of DNA used for digestion will be lesser as I will leave 1ul of it as backup (so I can PCR more) incase something goes wrong. But perhaps that is an okay compromise to speed things up. Thanks.
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