Single Stranded DNA Specific Stain

[Regina Shaw]

In article <326E6224.5BBC at vetsci.microvet.arizona.edu>,
billing at vetsci.microvet.arizona.edu wrote:

> Is anyone aware of a stain that is specific for single stranded DNA?  I 
> seem to recall something about acridine orange, but the ol' brain cells 
> can't be certain.
> 
> I am trying to find an easy method to detect single stranded 
> plasmid intermediates on agarose gels?  If not specific stains, any 
> other ideas?
> 
> TIA
> 
> Steve

Hi, Steve,

when viewed on a UV transilluminator (around 300 nm), acridine orange will
stain single stranded DNA bright orange-reddish, and double stranded DNA
yellowish green. The protocol is: (from USB T7-GEN in vitro mutagenesis
kit)
- run your gel (TAE or TBE)
- soak in a solution of 30 microgram/ml acridine orange in 10 mM sodium
phosphate pH 7.0, for 15-30 min at room temp (protect from light)
- destain very thoroughly, to reduce background, by several changes of HOT
tap water over 15-20 min. Works well for me...
Good luck,
Regina
Recombinant Protein Expression Core/ICBR