RNA extraction

~Jarvis~ nobody at nowhere.net
Wed Jul 19 11:18:29 EST 2000


What ill effects did the low ratio have on your Northerns?  

On Tue, 18 Jul 2000 16:36:49 -0400, "cracker" <besco.1 at osu.edu> wrote:

>I have found that if you use more Trizol in the initial extraction step,
>your ratios will improve.  The lower ratios seem to work fine for things
>such as RT-PCR, but, for me anyway, dont work as well in Northerns.....
>
>Neil Saunders <s3003334 at pop3.unsw.edu.au> wrote in message
>news:8ktl3v$atj$1 at mirv.comms.unsw.edu.au...
>> > I have isolated total RNA from non adherent cells with the TRIZOL
>reagent.
>> > At the end of the procedure, I solubilized the pellet with DEPC-treated
>> > water. After this, I measured OD 260 and 280 and calculated the ratio
>> > 260/280. This ratio should be 1.6 to 2.0. Sometimes I have got around
>1.6
>> > but generally 1.4 to 1.5  ... it is too low but the RNA is OK on agarose
>> gel
>> > (no apparent degradation). Which step of the Trizol method can I improve
>> to
>> > increase this ratio? I have already read that to calulate the ratio it
>is
>> > better to take ODs of RNA in TE buffer but other people have already got
>> > good ratios with RNA in water.
>>
>> If the RNA looks good on a gel, I wouldn't worry about the ratio.  What is
>> the source of the RNA?  In my experience with Gram -ve bacteria
>(Paracoccus
>> denitrificans and E. coli), it's normal for the ratio to vary between 1.5
>> and 1.8.  The theoretical ratio of 2 in all the manuals is just
>> that-theoretical.  It depends on the base composition and other factors eg
>> in some Gram -ves, the 23S cleaves into 2 fragments, but this isn't
>> indicative of general degradation.
>>
>> Neil
>>
>> --
>> School of Microbiology & Immunology,
>> University of New South Wales,
>> Sydney 2052,
>> Australia
>>
>> Ph: +61 2 9385 2093
>> Fx: +61 2 9385 1591
>> email: neil.saunders at unsw.edu.au
>> http://www.crosswinds.net/~nfws/index.htm
>> ArchaeaWeb
>> http://www.crosswinds.net/~nfws/archaea/index.htm
>>
>>
>>
>






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