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Gel electrophoresis and purifying dna


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#1 lyok

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Posted 22 August 2009 - 04:30 AM

Hallo ,

a little question from someone who hasnt worked a lot with Gel electrophoresis.

I am trying to extract some dna and then later on if its ok , I'll purify it and use it.
I however have the following question: should I do rnase treatment or not?
And how should I store this rnase anyway?
They used to make stock solutions here: freeze those and when needed thaw them, make a dilution of this stock solution and work with this one, but is it ok for the stock solution to be freezeed and thawed every time ? I cant imagine that this wouldnt affect the rnase?

thanks

Edited by lyok, 22 August 2009 - 08:50 AM.


#2 T C

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Posted 25 August 2009 - 02:36 AM

Hey,

Do RNase treatment and make single use aliquots and store at -80.

Best,
TC

#3 lyok

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Posted 27 August 2009 - 01:00 AM

Hey,

Do RNase treatment and make single use aliquots and store at -80.

Best,
TC


Yeah,

someone told me this too, but why?

What is the influence of this rna treatment then?

And you store the rnase in -80?

I was told to simply store it at -20 and everytime I needed it, I was told to make a work solution and work with that.
So in general: the rnase is stored at -20 and thawed every time I need it , and frozen again when I have my work solution.

Would the rnase activity go bad when freezing and thawing every time?

#4 T C

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Posted 28 August 2009 - 07:25 AM

Hey,

You can store it at -20 degrees and RNAse would be fine...I know of many labs which do so......... infact P1 RNase buffer is stored at 4 degrees.


RNase activity does go bad with freeze and thaw cycles......personal observation and don't know the real reason but maybe its so coz of wear and tear of the protein?

Best,
TC


Yeah,

someone told me this too, but why?

What is the influence of this rna treatment then?

And you store the rnase in -80?

I was told to simply store it at -20 and everytime I needed it, I was told to make a work solution and work with that.
So in general: the rnase is stored at -20 and thawed every time I need it , and frozen again when I have my work solution.

Would the rnase activity go bad when freezing and thawing every time?



#5 lyok

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Posted 31 August 2009 - 12:47 AM

Hey,

You can store it at -20 degrees and RNAse would be fine...I know of many labs which do so......... infact P1 RNase buffer is stored at 4 degrees.


RNase activity does go bad with freeze and thaw cycles......personal observation and don't know the real reason but maybe its so coz of wear and tear of the protein?

Best,
TC


Yeah, here at the lab they told me the same thing. That rnase treatment would give a better result on the gel, however they couldnt explain me why.
Seems that no one really knows why this is.


So, its not good to thaw and freeze... thats what I was thinking too, but they insist that I can do it like that (freeze and thaw it everytime).

#6 s_laub

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Posted 06 September 2009 - 10:29 PM

I just read off of wikipedia that RNase is incredibly stable and the reason that it is typically boiled before use is because you're provided with a crude extract that contains a variety of proteins that denature when boiled. RNase can withstand this treatment and after boiling it's suitable for use. So I'm guessing if it can withstand 100*C for sometime, you could probably freeze/thaw it without much worry.

#7 Warren

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Posted 07 September 2009 - 11:29 AM

I just read off of wikipedia that RNase is incredibly stable and the reason that it is typically boiled before use is because you're provided with a crude extract that contains a variety of proteins that denature when boiled. RNase can withstand this treatment and after boiling it's suitable for use. So I'm guessing if it can withstand 100*C for sometime, you could probably freeze/thaw it without much worry.


RNase A (which I believe is the one you are referring to) is indeed incredibly stable. It is one of the few enzymes used in molecular biology that you really don't have to worry about, and can forget everything you have learned about storing enzymes! You can store it at room temp if you want. I have never seen a decrease in activity from freeze/thawing, although this could potentially shear a molecule. You pretty much need phenol to destroy it. As long as nothing is growing in it, it will probably last forever (or at least your lifetime :blink: Warren..




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