the effect of tRNA on cDNA cloning ?

Ned Mantei mantei at neuro.biol.ethz.ch
Wed Dec 27 10:06:40 EST 1995


In article <4ao0p2$ahd at bubba.NMSU.Edu>, smori at nmsu.edu (Shahram Mori) wrote:

> Seongwook Kwon (swkwon at email.unc.edu) wrote:
> : Hi,
> 
> : I am thinking of using tRNA to help out precipitating my cDNAs.  These 
> : cDNAs are to be used for a library construction.
> 

> Or
> 
> 2) Add a little Rnase to the resuspended pellet.
> Cheers
> --


This is something that I would *not* do. (1) RNase is often contaminated
with DNase. Even if you heat it to inactivate DNase, it seems to slowly
renature during storage (or at least it's there again later!). Also, (2)
RNase sticks strongly to single-stranded DNA, especially if there is no
RNA around. Inhibition of the ligase reaction seems a real possibility.

In fact, RNA seems to be invisible to DNA ligase, and I have never seen
problems with precipitating cDNA with about 5 micrograms of RNA as
carrier.
Other possibilities are glycogen, as already suggested (I would use about
10 micrograms, not 50), or linear polyacrylamide (about 3--5 micrograms).

-- 
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046



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