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Lysis buffer


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9 replies to this topic

#1 Piglet

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Posted 25 February 2010 - 06:27 AM

Hi!

I'm trying to prepare 10X lysis buffer, but I have problems with solubility. The buffer should be 0,5 M EDTA; 0,5 M Tris; and 10 % SDS. I adjusted pH just above 8, but there is something that doesn't want to dissolve. Could you please help me!?

#2 laurequillo

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Posted 25 February 2010 - 08:17 AM

It is the SDS I guess. You should "warm" it to allow the SDS to solubilize
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#3 scolix

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Posted 25 February 2010 - 08:21 AM

i would also guess it to be SDS. warm it

#4 Piglet

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Posted 25 February 2010 - 11:02 PM

Yes, I tried also to warm it to 70-80 C... It didn't help. There solution is otherwise clear, but there is a thick precipitation at the top...

#5 laurequillo

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Posted 25 February 2010 - 11:45 PM

Yes, I tried also to warm it to 70-80 C... It didn't help. There solution is otherwise clear, but there is a thick precipitation at the top...


Why don you try to add the SDS to the final 1X solution? I know that a 1%SDS solution is ok, I never tried such amount of SDS.
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#6 Piglet

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Posted 26 February 2010 - 12:27 AM

Because I need to have 10x buffer to add to my samples... I have liquid samples, and if I add 1X buffer the samples would dilute too much. I think now I'll try to make more concentrated solutions of each ingredient, and combine them as liquids. Before I had just EDTA as a liquid.

#7 phage434

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Posted 26 February 2010 - 06:45 PM

If you have never made 0.5 M EDTA solution before, you should know that it won't go into solution at all until neutralized with NaOH pellets. Also, you can't make 1 M EDTA -- it is not sufficiently soluble even when neutralized.

Your life would be much simpler if you made a 5x buffer, using 0.5 M EDTA stock solution, 1 M Tris solution, and 20% SDS.

#8 Piglet

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Posted 01 March 2010 - 06:29 AM

But why should life be simple and without challenges :lol:

Thank you all for your comments, finally after a week in + 70 C my buffer seems to be clear!

#9 bob1

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Posted 01 March 2010 - 03:30 PM

10% SDS should be fine at room temp - it will ppt at about 18 deg C though. I suspect it is the EDTA precipitating, like it does in TBE, as it is near its solubility limits, the addition of ions or slight changes in pH will cause it to come out of solution.

#10 hanming86

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Posted 04 March 2010 - 08:25 AM

But why should life be simple and without challenges :(

Thank you all for your comments, finally after a week in + 70 C my buffer seems to be clear!


I also suspect it is EDTA. A week in 70C.. crazy haha.
however if at all time u could keep it simple, please do. Research is a race against time.
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