You need to have relevant stock solutions to start with, e.g., 1 M KCl, 1M MgCl2 etc.
Then, say you wanted to make 100 ml of solution with final conc. 10 mM KCl, 1 mM MgCl2.
You would add, 1 ml of 1 M KCl, and 0.1 ml of 1M MgCl2, and 98.9 ml of H2O. Same principle applies for Lysis Buffer.
For Protease K you should make up your stock in 50% glycerol and store at -20. It keeps better that way than when frozen in water.
I'm feeling a little dense here. I'm trying to do single worm PCR, but I... can't understand how to make the worm lysis buffer (listed in the first post).I’ve poked around the internet a bit, and everywhere lists the ingredients in mM and in %; I’m probably missing something, as I always thought that some kind of quantity or volume was useful in addition to the concentration in order to be able to mix up a desired solution.
If someone could explain or translate, I’d be most appreciative.
The molarities and percentages given are for the concentrations in the final solution. Handle each as you normally would, and don’t worry that different components have their representation described differently:
50 mM KCl
10 mM Tris pH 8.3
2.5 mM MgCl2
0.45% NP-40 (IGEPAL)
0.45% Tween-20
0.01% Gelatin
So, you make up stock concentrations of KCl, Tris pH 8.3, and MgCl2, each at least 10-fold higher concentration than you will have in the completed solution. In making the lysis buffer, you use the appropriate amount of each to reach the desired final concentration.
NP40 and Tween are detergents, in liquid form, so the concentration is volume per volume. Because they are viscous and difficult to pipet accurately undiluted, the easiest method is to decant some into a (graduated) 15ml conical and add an equal amount of water to get a 50% stock; mix well, and this can be pipetted accurately. Leftover 50% detergent can be saved at room temperature for a long time.
Gelatin is a solid, so should be handled as weight per volume.
I’ve sterile-filtered the solution after making it; I have my suspicions this may remove a lot of the gelatin, but the buffer works.
Note the above recipe is for 1x lysis buffer; some people prefer to make 2x lysis buffer, as they can then add equal volumes of it to worms in water, which you sometimes want to do. 1x lysis buffer is mostly useful for picking worms into; this is a very handy thing to be able to do (it’s served essentially all of my needs, and I make and use 1x buffer), but some people strongly prefer to make 2x buffer.
just a final note.
Hillel, my experience (from cell culture) is that filter sterilising gelatin solutions does not result in losses as long as the gelatin is completely dissolved. For higher % solutions (e.g. 0.1%) this is important and may need 60 degrees to get it dissolved. For the concentration here, that may not be as important.
But, as you say, the lysis buffer works…
Steve