Maxam-Gilbert Who?!

John j.mitchell at ic.ac.uk
Thu Nov 18 11:13:42 EST 1999


Dear Eric

The reason you have to use M+G sequencing is that you need to align the
ladder with you footprint base for base, you cannot do this using Sanger
sequencing due to the way in which the DNA is labelled as John Lye
suggested.  Therefore the best way is to use M+G sequencing even if it
does mean using lovely chemicals!

The reason you are no longer able to buy a M+G sequencing kit is thanks
to drugs.  Piperidine which is used to cleave the DNA molecule once it
has been  modified with the various chemicals to give you the ladder is
used in one of the steps in the purification of a class A drug (I cannot
think which one it is now).  This has resulted in the requirement of a
licence to buy the chemical (Standford should have one so you should not
have any trouble getting it) which forced NEN to stop producing the kit.

Maniatis has a good section on M+G sequencing chemistry and lays out the
method rather well (chapter 13.78-13.101).  You can buy (or at least
used to) from Amersham Hybond M+G membrane which allows you to anneal
the DNA you wish to sequence onto a membrane thereby allowing you to
wash your DNA  a little easier (I have never used it so I cannot tell
you how good it is).  It is quite a good idea if it is possible to find
a restriction site very close (>20bp) to the end of the DNA you do not
wish to sequence as this will make sure that the only sequence you see
at the end is from the end of the DNA molecule you wish to see.

If you want some more help write back

John

--
Dr J. Mitchell
Imperial College School of Medicine
Dept Neuromuscular Diseases
Rm 8L16 Lab Block
Charing Cross Campus
London
W6 8RP
Tel. 0181 3830519
Fax. 0181 8467099






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