AgNO3 DNA Staining Procedure Questions (David Atherton, are you there?)
901106c at axe.acadiau.ca
Thu Jun 17 16:28:05 EST 1993
I attempted to mail this letter to David Atherton, who posted the procedure
I attempted. However, the mail bounced many times, so I give up. Here is my
question. If anyone can help, I would GREATLY appreciate it!
I was wondering if you could help me out a bit: I recently saved the
protocol you gave in the molbio.reagents news group for DNA staining
that does not use EtBr (Sounds good to me!)
The procedure I followed was as per your directions. However, it has
been developing in the Na2CO3/Formamide soln' for well over 30
minutes now, and I am starting to wonder if I did something wrong. I
tried to check Maniatis, but alas our version is decades old (the one
volume version) and does not seem to contain the references I tried
to find for silver staining.
My attempt was on a mini gel (approx 10x15 cm) that I ran yesterday.
It had been stained with EtBr yesterday, but had soaked overnight in
many volumes of water. I then followed your protocol today.
The protocol I followed was:
-Wash gel 20 min in distilled water with shaking
-wash gel in 0.3% Ammonium Chloride solution
-Mix up: 75 ml 0.15% Silver Nitrate+0.1ml 5M NaOH --looked like
white coffee! --then added dropwise approx 500ul Ammonium Hydroxide -
--at this point, soln' did become clear, but had some pcp present...
is this unusual?)
-washed for 15 min in above soln'
-Rinsed in several volumes distilled water 3 times.
-added stain soln (2% Na2CO3 + 0.2% Formamide
It is still sitting there. I would love to get this procedure working
if I can.
I am aware that I made some assumptions about reagents in some places.
If I am making incorrect assumptions or have any obvious holes in my
procedure, please let me know...
Thanks very much!
........ Tim Chipman ..................................
.......... 901106c at axe.acadiau.ca ..................................
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