dsDNA sequencing protocol

Sasha Kraev 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 
twice.
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
quality
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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