Temperatures for precipitation of DNA

CK Wen cwen at aux.btny.purdue.edu
Sat Jan 20 10:35:49 EST 1996


nswema05 at angis.su.OZ.AU wrote:
>
>When I started working in a molecular biology laboratory, it was
>recommended that I hold DNA at -20oC for maximal yields when
>precipitating with NaCl in EtOH. However, I have noted that many
>protocols advise holding DNA for precipitation at room temperature.
>Also, when using NH4SO4 in EtOH for precipitation of DNA, some
>protocols state that yields may be reduced at lower temperatures.
>When using propan-1-ol, most protocols recommend precipitation at
>room temperature.
>
>What are the optimal temperatures for precipitation of DNA using
>various combinations of salts and alcohols?
>
>Adrian W Philbey
>Elizabeth Macarthur Agricultural Institute
>Camden, New South Wales
>Australia
>
Dear Adrian:

  I always precipitate DNA/RNA on ice for 30min to 1hr and get good 
recovery. For example, isolating DNA/RNA from plant cells, 500ug of 
DNA/RNA in a total vol of 10ml of NaOAc/EtOH precipitation can be 
recoveried, which means that the conc is as low as 0.5ug/ml of DNA/RNA. 

  What I think is that at higher temperature, on ice rathen than -20C, 
the
molecules or particles have higher energy to move(called as Brown 
movement??? I am not sure), and therefore, the chance of collision 
between two particles is increased. Totally, particles collide to each
other and aggregate with each other to form bigger particles which have
heavier weight to precipitate.  DNA/RNA should become insoluble forms
once NaOAc/EtOH is added, however, if the size of aggregated particles
were not big enough, either precipitation efficiency is decreased, or 
the ppt is spreaded on the wall of the tube and get off easily after
70% EtOH wash, and the resulting DNA/RNA recovery is decreased.  For the 
-20C/overnight procedure, I would like to say that an overnight stand
could let most of the small particles to set at the bottom of the tube, 
not increasing the efficiency of EtOH ppt.

 For the NH4OAc/EtOH procedure, my experience tells me that this 
procedure gives me purer DNA/RNA, but I do not really not why.

regard,

Chi-Kuang





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