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rnase treatment


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#1 epigenetics

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Posted 21 July 2010 - 02:19 PM

Hello all,

I did a stupid mistake today and am thinking what to do except waiting for the gel pic what i will get after 2 days.
I use phenol chloroform to extract DNA from cells. So i treat the cells with extraction buffer and it has rnase a. After that i treat with proteinase K and leave overnight. But today instead of adding proteinase K, i added Rnase A again and now conc of rnase a is really really high in my sample. I talked to Sigma about the inactivation temp of rnase a, but they dont know.

So my question is, how can high conc of rnase a affect ds DNA provided Rnase A is Dnase free?

Any help would be appreciated.

thanks,

#2 perneseblue

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Posted 21 July 2010 - 04:56 PM

RNAse A cannot be heat inactivated! It survives autoclaving rather well. And the method to prepare RNAse requires you to boil the sample for 15minutes.

Provided the RNAse is DNAse free, excess RNAse will not have any effect on your DNA sample. You might want to remove this RNAse if you are going to transform/transfect your DNA sample into cells.
May your PCR products be long, your protocols short and your boss on holiday

#3 Curtis

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Posted 23 July 2010 - 03:34 AM

perneseblue is right. you can simply purify your DNA sample by running on gel or column purification. you probably have a lot of DNA after extraction to not worry about concentration. why wait for sigma for days to receive a reply?




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