Looking to experiment ways to speed up the process of protein digestion for nucleic acid purification using Proteinase K. Currently our lysis buffer includes low concentration SDS and EDTA but not urea. Would it be beneficial to add urea to this buffer? I understand based off info from Sigma-Aldrich's website that addition of EDTA reduces catalytic activity of PK by 25% by reallocating Ca2+ ions which PK requires for activation. Based off these details I am led to question if a better protocol would include exposure of the sample first to SDS/urea prep buffer, then second, to PK, and finally EDTA, effectively giving the PK some time to digest at 100% strength before 25% reduction by involving EDTA, instead of all 3 simultaneously. I understand the purpose of the EDTA addition is to regulate the acidity and osmolarity of the lysate and I am also wondering if the above stated adjustment would put the sample in too much risk of high acidity and osmolarity. Finally, our current incubation procedure (incubating overnight at 50-60 degrees Celsius) is working very well but I'd like to reduce the time if possible at all. I'd greatly appreciate any input on ways to incubate this mixture for less time while still getting optimal results, or ways anybody has found to reduce this process in general. Thank you very much.
Dear Matthew
- i have done what you have suggested many times
- Firstly do not add urea to your initial lysate mixture. This will denature proteinase K which is a crucial component of the digestion/lysis mix
- In essence SDS breaks open the cells and prot k digests away residual protein
- Thus digest with SDS and proteinase K in either standard TE or TNE where N is sodium chloride: TE will work fine
- Regarding the temp and shaving time off your digestion time it is routine to digest over night at 37C and 50-55C for 1 hour. Thus try your current temp and 1 hour should suffice
Dear Matthew
- i have done what you have suggested many times
- Firstly do not add urea to your initial lysate mixture. This will denature proteinase K which is a crucial component of the digestion/lysis mix
- In essence SDS breaks open the cells and prot k digests away residual protein
- Thus digest with SDS and proteinase K in either standard TE or TNE where N is sodium chloride: TE will work fine
- Regarding the temp and shaving time off your digestion time it is routine to digest over night at 37C and 50-55C for 1 hour. Thus try your current temp and 1 hour should suffice
Hi Matthew
A final concentration of EDTA up to 40mM in your lysis buffer will no affect the PK activity In my case the mixture TNE as quoted by Laurence above plus SDS 4% final concentration works very good.
In reference to the temperature 55°C for 1 hour is enough but it will also depend of the amount of the pellet (asuming bacterial pellet) you have. You will notice if a good digestion accurs when the viscosity of your lysate mixture increase.
I concur with comments by Laurence and Wilbert. Incubating 1 h at 55C with proteinase K and SDS should work well. To speed up the reaction further you could try adding more proteinase K and use as high as possible level of SDS that won't disturb your downstream applications. If you heat inactivate proteinase K, avoid high temperatures to avoid DNA fragmentation. 85C for 20 min is sufficient for inactivation. Salts will also protect DNA during heat inactivation, so add NaCl or PBS before the inactivation step. I have noticed that even a small addition (5-10% volume) of PBS helps to keep DNA intact during heat inactivation.
Dear Lee. All Proteinase K products I can think of come with inserts stating 8.0 is, if not the optomal pH, at least within the optimum range. Some brands say working range pH is 4-12, some say 4-12.5 or 7.5-12. So pH 8 should always work. If you need to use a different pH for your application it is good to know that there are slight differences between brands with regard to pH optimum.
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