Lysis buffer protocols

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haileyp
haileyp's picture
Lysis buffer protocols

I work in a molecular ecology lab and my boss has given me some White Tailed Deer lymph node samples from which to extract DNA. The protocol that I'm using requires me to make a 1x Lysis buffer. The recipe that was recommended to me looks like this:
1.21g Tris base
37.22g EDTA
5.00g SDS
All of this in 1 L of water
But I have not been able to get this mixture to go into solution with constant stirring with a stir bar, or with heating. If anyone has any suggestions for a way to keep all of this in solution or has another lysis buffer to recommend I would appreciate it!

PhilM
PhilM's picture
If your lab can afford it use
Ivan Delgado
Ivan Delgado's picture
 

 
Hi haileyp,
Your problem in getting your lysis buffer into solution may be due to the way in which you are preparing it. First of all I would recommend preparing stock solutions first and not trying to get all these solids into solution together. While getting Tris into solution is not hard, SDS and especially EDTA can be a problem. For example EDTA will not go into solution unless it is close to pH 8, which is why it is always recommended to prepare a 0.5 M EDTA solution (pH 8 with solid NaOH) and then using this solution to prepare other solutions.
Hope this helps

Fraser Moss
Fraser Moss's picture
These days you can buy stock

These days you can buy stock solutions of most of these components from most of the major reagent suppliers (Sigma, Fisher Fluka etc).  With the time you save not weighing out powders etc you more than compensate the apparent higher cost of buying the solution premade.

Ivan Delgado
Ivan Delgado's picture
 

 
I agree 100% with frasermoss. 100 ml of 0.5 M EDTA only costs about $20 and you do not have to worry about anything not working (like the pH meter, the balance, etc). I do think though that you should try to keep up with why things work the way they do. In other words, as you buy already prepared solutions and kits to perform your research, try to learn the reasons why these reagents work the way they do. For example having an appreciation for the basic biochemical characteristics of buffers, and how they work under different pH conditions and the like, can only help you in the long run.

g a
g a's picture
Just like other friends who

Just like other friends who have already posted I also expect that its EDTA which is sparingly soluble unless its at pH8. however with Tris the pH gets more towards the basic range exceeding even 8 so why the EDTA still is insoluble i have absolutely no IDEA......... although it is well known that EDTA is solubilized using NaOH but then the requirement is of pH and not of NaOH.
 
may be you can shed more light yourself by informing us in which order did you added the reagents?
Cheers
Gaganjot Singh