DNAse treatment of isolated total RNA ?

John Chesters jkc at rri.sari.ac.uk
Fri Jan 26 07:54:08 EST 1996


Daniel Pereira <mgs6145 at student.health.ufl.edu> wrote:
>T.Jensen at immi.ku.dk (Teis Jensen) wrote:
>>
>> I am using RT-PCR in my work but I have problems with small traces of DNA
>> contamination in my RNA samples. I am following the acid-phenol method for
>> isolation of total RNA but would like to include a DNAse treatment step
>> during the isolation procedure. However, how should I do this. Should I
>> treat the RNA during of after the final EtOH precipitation in the isolation
>> procedure and is it necessary to remove the DNAse afterwords with
>> phenol:chloroform (or just a temperature denaturation) ?
>> 
>> Hope to hear from you
>> 
>> Teis
>> __________________________________________________________________
>> Teis Jensen
>> INSTITUTE OF MEDICAL MICROBIOLOGY
>> AND IMMUNOLOGY
>> University of Copenhagen
>> Panum Institute; 22.5
>> Blegdamsvej 3C
>> Dk-2200 Kbh.N
>> 
>> Tlf:(+45)35327867
>> Fax:(+45)35327851
>> __________________________________________________________________
>> 
>> It is quite simple to get rid of contaminating DNA. I use the following
>protocol to do this.
>From the isopropanol suspension- Spin down 10 minutes
>wash pellet in 70% EtOH, air dry
>resuspend pellet in 50 ul TE and 50ul DNase digestion B (10mM MgCl2, 1mM DTT,2U DNase 1--Worthington Biochem
>4U RNasin) 
>incubate for 15 min at 37
>add 25 ul DNase stop solution ( 50mM EDTA, 1.5M Sodium Acetate, 1% SDS)
>phenol:chloroform extract 
>Add 2 volumes of EtOH
>Then your ready to go
>Good luck





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