PCR to Agarose Gel Shortcuts
jladasky at pmgm.Stanford.EDU
Thu Oct 22 17:08:20 EST 1998
In article <362F8BC9.69BA63BE at sph.unc.edu>,
John Scott Meschke <jmeschke at sph.unc.edu> wrote:
>Does anyone having time saving shortcuts for running PCR products on an
>My current protocol involves aliquoting 15 microliters (of 100) of
>product with 3 microliters of 1x gibco loading buffer. I am considering
>skipping the aliquot step and putting loading buffer directly into my
>product tube. This will eliminate a centrifuge step, and at least one
>pippetting step. What I am concerned about is the stability of the cDNA
>in the presence of loading buffer over time. The assays I am running
>are merely presence absence and I do not need to manipulate the DNA
>after running the gel except for gene probing for confirmation. Does
>this sound like a good idea? Are there any other time savers out
>there? I have considered red taq, but I am concerned with the potential
>differences between the Amplitaq that I currently use and the red taq.
>My experiments are long term comparisons and I hate to modify my
>protocols too much over the course of the experiment. Thanks alot.
I have been using a combination PCR and loading buffer for some
time now. There was a Biotechniques article about PCR-compatible load-
ing buffer components a few years back. Sorry, I'm not in front of my
references at the moment. Anyway, you can make up 25 ul PCR's contain-
ing sucrose, cresol red, and tartrazine yellow, and go straight from
the tube to the gel. I have run several samples in parallel, with and
without the loading dye components. I see little difference, though I
have only tried PCR's under 1 kB, with Taq polymerase only.
If there is interest, I will post or email my buffer recipe,
when I get to my references.
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw. - John Ladasky
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