[Zbrafish] Problems with DNAse

Burdine, Rebecca D via zbrafish%40net.bio.net (by rburdine from Princeton.EDU)
Fri May 29 12:59:23 EST 2009


Hi,

TRIZOL uses guanidine isothiocyanate and acid phenol to isolate RNA based on the original paper by Chomczynski, P. and Sacchi, N. Anal. Biochem 162, 156 (1987).  The acid phenol step should partition the RNA away from the DNA so you shouldn't need to DNAse if your preps are clean and you avoided the interface when separating the aqueous solution from the phenol.

However, you shouldn't lose your RNA when you use DNAse regardless.  Are you sure it is an RNAse free DNAse?  You may want to purchase a new vial and give that a try.

Becky

---------------------------------------------------
Rebecca D. Burdine, Ph.D.
Assistant Professor
Dept. of Molecular Biology
Princeton University
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> -----Original Message-----
> From: zbrafish-bounces from oat.bio.indiana.edu [mailto:zbrafish-
> bounces from oat.bio.indiana.edu] On Behalf Of Usua
> Sent: Thursday, May 28, 2009 10:02 AM
> To: bionet-organisms-zebrafish from moderators.isc.org
> Subject: [Zbrafish] Problems with DNAse
> 
> Dear all,
> 
> I am working with zebrafish embryos and gene expression. I use trizol
> for the RNA extraction, and then when I use DNAse before the RT PCR, I
> lose all the RNA in my sample.
> 
> does anyone use a DNAse step in their RNA preps?
> 
> I would like to know if it is neccessary to use dnases or if I could
> play directly the PCR.  I know that my preps theoretically should not
> yield DNA product, but i want to be sure that the RTPCR products I am
> recieving are from an RNA template and not from DNA.
> 
> Thank you in advance
> .
> 
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