Last week I posted a series of questions concerning Tc1 STS mapping here.
I would like to thank everybody who gave me helpful answers and comments.
Even more people emailed me to express their interest in the answers to the
questions I posted. Here is a summary of the comments I got.
1. Is there a common source for the primers now?
Apparently not. Therefore as follow-up question: does anybody know of a
cheap source for the primers? The company we are using now offers 0.95$ a
base on a 100nmol scale (1.15$ for 200 nmole).
2. Has anyone extended the original set of markers provided by Ben Williams
and coworkers, e.g. included some of the newly mapped Tc1 sites from the
The set of markers (mapped Tc1 insertions) certainly is being extended
(Plasterk and Thaden labs), and people made and tested primers for some of
the new insertions but if I understood correctly nobody so far created a
set(s) of primers for multiplex PCR like Ben Williams et al. did, or
expanded Ben's original set.
3. Is the map position of the markers in the methods books up to date or
has the position of any of the markers changed since then. (Are the
insertion points in Acedb? I couldn't find them.)
Map positions seem up to date. If anybody finds inconsistencies in the
positions, maybe he or she could make the information available in the
newsgroup. The Tc1 insertions are of course in Acedb. However, the set Ben
used is in class 'locus' and not in class 'Tc1 insertion' where I looked.
4. Has anyone changes/improvements for the procedure?
Ron Plasterk wrote: We work on a display (cpf van Luenen's last WBG
article), which may get rid of all primer-making to visualize the Tc1
Out of curiosity: Why are the markers spaced so closely that you need to
run a acrylamid gel to separate them?
John Thaden wrote: I think because the sequencing runs done by Williams et
al. were short. That, and the usual sequence constraints one has when
designing well-behaved PCR primers.
Several people pointed out alternatives to acrylamid gels for separating
the fragments. They apparently separate quite nicely on
a) 3:1 NuSieve (FMC):SeaKem (FMC) agarose gels with 4% (w/v) total agarose
b) 6% Nusieve agarose from FMC
c) even on an agarose gel (most of the bands), as long as you make it very
thin (eg pour agaorse on a small plate, don't use a tray).
Harald Hutter Hedgecock lab
Johns Hopkins University
Department of Biology
Room 7, Mudd Hall phone +1 410 516 8381
3400 N. Charles Street fax +1 410 516 5213
Baltimore, MD 21218, USA email hutter at jhuvms.hcf.jhu.edu