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How to Make Nucleotide Ladder Markers?

How to Make Nucleotide Ladder Markers? - Protocols and Methods Forum

How to Make Nucleotide Ladder Markers? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 11-25-2004, 06:20 PM
Bert Gold
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Default How to Make Nucleotide Ladder Markers?



Dear netters,

I need to make markers to reduce costs
and improve quality of STR detection in
my lab.

I can probably make a fluorescent labeled
oligo to prime in one direction with,
but, I need ideas for how to make a
mix containing specifically sized
products.

How would you make a ladder that
consists of: 60, 70, 80, 90, 100,
120, 140, 160, 180, 190, 200, 220,
240, 260, 280, 300, 320, 340, 360,
380, 400, 420, 440, 460, 480, 500,
520, 540, 560, 580, 600, 620, and
640 nucleotides?

Of course, I can purchase some
pre-existing ladders, if you think
they will help.

Please provide me your best thoughts,
and I will air them here (or in some
other public space).

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  #2  
Old 11-26-2004, 10:38 AM
Duncan Clark
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Default How to Make Nucleotide Ladder Markers?

Historians believe that in newspost <[Only registered users see links. ]> on
Thu, 25 Nov 2004, Bert Gold <[Only registered users see links. ]> penned the
following literary masterpiece:

Umpteen individual PCRs - watch out for the +A addition?

Or mutate or gene synthesise a DNA that has say C only at those
positions. Use a single fluorescent ddGTP sequencing reaction so only
those positions light up?

In the latter case you can't use a fluorescent primer as you will
generate a fluorescent run-off product much much brighter than the
fragments you require.

Duncan

--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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  #3  
Old 11-27-2004, 12:13 AM
Bert Gold
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Default How to Make Nucleotide Ladder Markers?

After Duncan posted his reply, I found:
DeWoody JA. Schupp J. Kenefic L. Busch J. Murfitt L. Keim P. Universal
method for producing ROX-labeled size standards suitable for automated
genotyping. [Article] /Biotechniques. 37(3):348-+, 2004 Sep.

and

[Only registered users see links. ]

I'm still not satisfied that I've gotten to the best
approach, however.

I am thinking about some ligase based method.

Anyhow, I am still open to ideas.

In article <[Only registered users see links. ].com>, Duncan Clark says...

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