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dna storage

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dna storage

Postby hara » Tue Jan 09, 2007 2:13 pm

when we complete the isolation of dna from leukocytes and before we do spectrophotometry to check the ratio and the concetration of it is it o.k to dilute it in depc water even if the ph of it is acidic?
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Postby canalon » Tue Jan 09, 2007 7:10 pm

As a rule Tris pH 8 or TE 10mM are better if they do not interfere with the following experiments. But if it is just for the reading and you will discard your sample afterwards, go with water.
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Postby hara » Wed Jan 10, 2007 9:22 am

thank you..but i worry that the EDTA (in tris) will cause problems later to my pcr as it chelates the divalent cations of Mg+2...but when i use depc H2O i have a difficulty in diluting my dna as it is acidic and the dna is diluting in slightly alcalic pH...THANK YOU FOR THE REPLY
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Postby canalon » Wed Jan 10, 2007 3:19 pm

TE 10mM once diluted 1:25 or 1:50 in a PCR should not significantly alter conditions. but as I say, if you have to use less diluted solutions, I suggest simply Tris pH 8.0 (the elution buffer in many DNA purifications kits) this should be OK and avoid EDTA downstream.
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Postby hara » Fri Jan 12, 2007 12:33 pm

when you say tris pH=8.0 you mean to dissolve tris base in distilled water and then adjust the pH to 8.0 with HCl and sterilize by autoclaving?is this the solution you mean?thank you
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Postby canalon » Fri Jan 12, 2007 1:34 pm

yes. In the sigma catalog you can even find a table telling you exactly how much tris base and tris HCl you have to mix together to have a wide range of pH. (at tris or Trizma)
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Postby MrMistery » Fri Jan 12, 2007 1:39 pm

lol, what you guys just discussed looks like chineese to me...
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Postby canalon » Fri Jan 12, 2007 1:43 pm

One day you will find those things are just the base of what you need to know to use molecular biology.

By the way Tris is a buffer, which exist in 2 form (base and HCl) and hence have to pKa. So depending of the ratio of the 2 forms you can change the buffering pH of your solution.

EDTA is a cations chelating agent, it helps protect DNA from DNAses which needs Ca++ to act, but it can hinder Dna polymerase who needs Mg++ to work in later reactions.
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Postby SororSaudade » Fri Jan 12, 2007 4:57 pm

if EDTA is in high concentrations so that it interfers with your PCR reaction you can just simply increase the Mg concentration in the PCR mix. any standard PCR protocol should mention that.
I had some problems with kits for dna purification when using water due to diferences in pH.
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Postby canalon » Fri Jan 12, 2007 6:52 pm

SororSaudade wrote:if EDTA is in high concentrations so that it interfers with your PCR reaction you can just simply increase the Mg concentration in the PCR mix. any standard PCR protocol should mention that.


True, but why bother having to validate your conditions again if you can avoid it. A tris solution with a pH between 7 and 8 works well in most of the case.

SororSaudade wrote:I had some problems with kits for dna purification when using water due to diferences in pH.


They usually provide an elution buffer that is in most case Tris 10mM at the optimal pH for the column.
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Postby SororSaudade » Sat Jan 13, 2007 2:58 pm

what I ment is that I prefer to use buffers (in this case tris pH=8 ) to avoid problems in the next steps.
And I also pointed out that EDTA might not be such a huge problem.
I guess it also depends on how you're used to work... it was just my opinion :)
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