dissolving of RNA

Frank Maiwald maiwald at mpiz-koeln.mpg.de
Tue Mar 25 06:31:31 EST 1997


Thanks for every hint on my question !

Meanwhile my RNA dissolves just fine. I followed my original RNA
preparation protocoll (Logemann et al. 1987, Anal. Biochem. 163: 16-20)
increasing the number of phenol extractions (from four to six) using a
new batch of phenol. Additionally, I washed the nucleic acid pellet
after the acetic acid / EtOH precipitation twice with 3 M NaAc pH 5.5
and once with 70% EtOH. Then I resuspended the pellet in RNA extraction
buffer (containing 8 M guanidinium hydrochloride) and precipitated it
again with one vol. of 4 M LiCl at 4 C. After that I washed the pellet
twice with 70% EtOH and removed all traces of the EtOH with a pipette.
The resulting RNA pellets could be dissolved in 300 microliters of
DEPC-H2O within ten minutes.

As the increase of phenol extractions and washing steps resolved the
problem I think that contamination with (glyco)proteins and possibly
salt were the causes.

					Frank



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