Aurintricarboxylic Acid/RNA

[pgegen at rnaworld.bio.ukans.edu]

In <47k9eo$eeb at newsstand.cit.cornell.edu>, Luis Destefano Beltran <Luis_Destefano at cornell.edu> writes:
>Dear netters:
>
>I am isolating total RNA from apple fruits. The extraction buffer 
>includes Aurintricarboxylic Acid (ATA). The final total RNA pellet is 
>pink. The red book says that this is normal since ATA binds RNA (?). I 
>am planning to go on with mRNA isolation and subsequently with cDNA 
>library construction My question is: will ATA affect these two steps, if 
>yes, how? and what can I do to prevent it? Please answer to 
>Luis_Destefano at cornell.edu
>Thanks in advance for your help

ATA will inhibit most enzymes that bind to nucleic acids; that's why its so effective as a nuclease inhibitor. It doesn't bind to RNA (they are both anions), but it does precipitate with RNA -- i.e., its solubility properties are similar. The classsical way to remove it is to pass the RNA over a small column of Sephadex (say, G-25) in sterile TE buffer. I've done this a few times and it didn't give complete separation, so I'd recommend a column with a 10-20:1 height:diameter ratio (say, 5 cm x 0.25cm). 

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