Cell Lysis Buffer Prep

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haileyp
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Cell Lysis Buffer Prep

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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:

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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!
 
 
 

g a
g a's picture
EDTA most probably was not

EDTA most probably was not dissolved in your solution. dissolve EDTA with NaOH pellets as it solubilizes only at pH 8.0. and then prepare your buffer.
cheers.
Gaganjot Singh

Jason King
Jason King's picture
I think most people will have

I think most people will have made up a 0.5M EDTA solution and then just take what they need. Remember, you need a LOT of NaOH pellets to disolve the EDTA to 0.5M, so if you are using a 500mL bottle, don't put more than about 200-250mL water to start with otherwise your volume will end up too high!

I would also make up a 10% SDS solution and in the same way just use as much as you need to make up the amount of lysis buffer you need. Do you really need to make up 1 litre.

In fact, I think I'd also have a 1M Tris buffer pH 7-8 as you need it. Then just make up about 20 mL of lysis buffer.

g a
g a's picture
I agree with parvoman.....

I agree with parvoman..... Its a regular practice we follow...... prepare a high conc stock solution for almost everything and then prepare the working solution right when you need to use.
Cheers
Gaganjot Singh