jladasky at cmgm.Stanford.EDU
Tue Mar 17 06:16:18 EST 1998
In article <350E4A1E at msm.cgnet.com>,
David Brayford <D.BRAYFORD at cabi.org> wrote:
>Can anyone please advise on appropriate concentrations of cresol red and
>sucrose to include in PCR reactions, in order to facilitate gel loading?
> Following published protocols I tried 0.2mM cresol red and 12% sucrose
>(final conc.) but got no amplification and no dye visible on the gel.
> Controls were lovely. I guess the sucrose is the problem. Any advice
>d.brayford at cabi.org
Wow. That's WAY too much sucrose. Maybe there's a decimal point
missing there? My 20X PCR-compatible loading dye recipe is 30% sucrose,
0.30% tartrazine yellow (optional), 0.15% cresol red. So the final con-
centration of sucrose in my PCR is 1.5%. I prepare the buffer in UV-irrad-
iated sterile water, but then I autoclave it again anyway. Call me para-
noid, but it works!
Based on some old loading buffer recipes in Maniatis, I started
using this as a 6X solution, but I found that PCR was altered and some-
times inhibited at that high concentration. I tried using less. Even at
a 20-fold dilution, the sucrose was still perfectly able to perform the
necessary function of getting the sample to the bottom of the well.
Has anyone tried this buffer with other reactions that one might
do a lot -- say, restriction digests?
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw. - John Ladasky
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