Questions on the RNA extraction

eric anderson e-anderson at ski.mskcc.org
Wed Dec 11 10:59:14 EST 1996


In article <01ICWC51ONYA90NGSM at usthk.ust.hk>, REGCHAK at USTHK.UST.HK wrote:

> Hi all,
>      I did a total RNA extraction on plant tissue by using the Guanidinium 
> Method as usual.  However, when I finished all the preciptation and washing
> step, I cann't dissolve all my suspected RNA pellet in the 400ul DEPC
> treated water even under 65oC for half an hour. I am sure that 170mg leaves
> should not cause such yield of RNA.  What's wrong in my RNA pellet?  And I
> don't whether too dry of the pellet will cause the loss of the yield?
> 

regina,

it's possible that you did just overdry your pellet and that's why you're
having a hard time resuspending it.  it's also possible that there is some
sort of contaminant somewhere along the line in the prep.

i think that the easiest thing to do is just to take the "pellet" and
re-precipitate it with isopropanol.  just add 0.7 vol of isopropanol and
then repeat the precipitation and EtOH washes.  then remove as much of the
70% EtOH as you can with a pipet tip and let it dry upside down on the
bench for 10-15 minutes, no more and not under vacuum.  there will
probably be some residual dampness in the tube but this is probably just
water and doesn't cause a problem (at least in my hands).  then do the
resuspension and see what happens.

the other thing that you can try is just to increase the volume until the
pellet does go into solution and *then* precipitate it again.

good luck,

eric

-- 
Eric C. Anderson
Memorial Sloan-Kettering Cancer Center
Sloan-Kettering Institute
1275 York Ave. Box 470
New York, NY  10028
(212) 639-2977
e-anderson at ski.mskcc.org



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