I'd like to prepare a Lysozyme and Lysozyme Digestion Buffer (25mM Tris-HCL, pH 8.0, 2.5mM EDTA, 1% Triton X-100) for bacterial cell lysate with a final Lysozyme concentration of 20mg/mL. I need to prepare 5mL of the buffer. I have some questions about the process:
1) Can I add Triton X-100 directly to the buffer, or do I have to dilute with PBS first?
2) Do I need to autoclave the buffer I prepare?
3) Would vortexing be enough for Lysozyme to dissolve in the buffer?
Thanks in advance.
1) If you are going to use 100% Triton X-100, it will be difficult to add the necessary amount (50 µl) accurately because the detergent is so viscous. Make or buy a 10% stock solution in water and add 500 µl. There is no need to use PBS.
2) No. If you want to store it, put it in the freezer. To use it, thaw it out and mix it thoroughly.
3) Yes, but be gentle about it, or else you will denature the protein.
Adam B Shapiro thank you so much, your answer really helped. The original manual's descriptions are vague and there are very little info out there about this.
Sincerely.
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