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How do I make GTE buffer for alkaline lysis?

alkaline lysis PCR plasmid isolation GTE buffer E.coli

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#1 MarieJones2291

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Posted 30 November 2013 - 12:47 PM

Hi! 
I need some help. I'm not sure how to make GTE buffer for alkaline lysis.
I need GTE (pH 8.0) made up of: 50 mM glucose, 25 mM Tris, 10 mM EDTA. I guess 100 ml of this buffer will be sufficient so let's assume I will be preparing 100 ml. And here's my question- how do I do it? I think I need to prepare stock solutions for each constituent: 1M glucose stock sol., 1M Tris and 0.5 M EDTA. And then I have to achieve final concentrations of each. So according to my calculations it would be as follows: 5ml of glucose, 2.5ml of Tris and 2 ml of EDTA in 100 ml. 
Do I mix all of them together and then bring up the volume to 100 ml with distilled water? Or do I weight out certain amount of each component (calculating first the amounts needed for 100 ml to achieve desired concentrations), add it all together and then add water to make 100 ml of total solution? Additionally, EDTA doesn't dissolve in water (until pH's reached 8.0 or above I guess) so can I add sodium hydroxide to it? 
If you could explain to me how to prepare this buffer, I would be very thankful. I know it's not a rocket science but really not sure how to do it. And I did all my calculations using following formula: C1 x V1 = C2 x V2.  Thanks in advance!
 

#2 phage434

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Posted 30 November 2013 - 12:58 PM

Your calculations are correct. If you are making Tris stock, you need to adjust the pH with hydrochloric acid (typically) to 8.0. An alternative is to prepare 1 M Tris base and 1 M Tris hydrochloride solutions and mix them together until the pH is correct. This has the advantage of never overshooting the correct pH, and always leaving the final solution at 1 M.

 

In making your EDTA stock, you will find the EDTA is insoluble until you add sufficient NaOH (use pellets until you are close).



#3 MarieJones2291

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Posted 09 December 2013 - 05:00 PM

Thanks!







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