dsDNA sequencing protocol
bckraev at aeolus.ethz.ch
Tue Nov 29 14:08:17 EST 1994
Jaz Baker asked for a plasmid sequencing protocol.
I am on the Net for only 3 months, but this question was already discussed
Paul, just when something gets into the FAQ list?
However, there are a number of basic points here.
1.You have a choice between cycle and non-cycle sequencing (CS is a FAQ item)
2.In non-cycle sequencing, the chances of getting a good result depend on two
factors: a) E.coli strain/growth time, b)purification procedure used
3.In any of the two seq procedures above, you have a choice of labelled primer
or internal labelling. The former somewhat compensates for inferior plasmid
4.In cycle sequencing, the strain choice applies, but the purification
procedure is less critical. Nice bands can be obtained from crude lysed
colony with a labeled primer protocol.
5.If you want it fast (and hot), Kodak BioMax X-ray film gives almost the
same sharp bands from 32P, as any regular film does for 35S or 33P(but
you need longer exposures).
The choice (e.g. a protocol) is yours. Some combinations (e.g. cycle sequencing
of column purified plasmid with Sequitherm and 33P labeled primers) do rival
M13 quality. Sequenase is usually OK (that means a 0-3 stops within a kilobase)
but sometimes is just a nightmare. Current instrumentation is just not good
enough to use only cycle sequencing. Hope that helps. Sasha
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