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Sequencing large banks

Alexander Kraev kraev at bc.biol.ethz.ch
Mon Oct 28 06:48:23 EST 1996

In article <abeeser-2710961524320001 at tcooper2.utmem.edu>,
abeeser at utmem1.utmem.edu (Sandy Beeser) wrote:

>   I am not sure I understand this, in the tecnical support from promega
> they suggest not sequencing lambda clones by direct incorportation.  I
> asked whether this was due to size or some property of lambda.  They said
> that it was due to lambda's secondary structure, not the size of lambda.

I routinely sequenced  150 ng of lambda DNA ( 5 fmol), using primers with
a Tm of
60 C or higher.  Before I started using long primers, I almost always got
pictures that were only visible after a Phosphorimager exposure for 2 days
and surely
could not be seen on film.  Using the right primer may improve results as
much as
100 fold in terms of band intensity. I do not understand in my turn what
it has to do
with the lambda secondary structure, because direct incorporation with 35S
is simply less
sensitive than using a 33P labelled primer. Besides that 500 fmol of
lambda  will not 
dissolve in the reaction volume.

> From this I assumed that 500fmol of a 3-5 kb plasmid was as sequenceable
> as 500 fmol of a 25 kb plasmid,

That is absolutely correct, also for a 200 kb BAC plasmid, only in the
latter case
dissolving 500 fmol in 10 ul might be difficult, to put it mildly.

> although you would have to add 8.3 micrograms of the 25 kb plasmid. 
Seeing as 
> I don't know the exact size of my plasmids my calculations say that it
> should be between 5.6 and 10.6 micrograms to get 500 fmol of 17-32 kb
> plasmids. 
Which brings you to using a DNA solution of 1 mg/ml in a sequencing
protocol. Any
impurity in this prep may shut up the polymerase reaction. With the labelled
primer you may start with 50 fmol ( or less if you accept long exposures)
 and that is one of the reasons why labelled primers generally work better with
less than perfectly pure DNA.

> I used this much and still got no sequence although as I said
> 322 sequenced fine at 500 fmol. 

Do you mean pBR322? Yes that much DNA sequences fine even on fluorescent
sequencers,  with either labelled primer or labelled terminators, however with
33P labelled primer or terminators you can do it from 50-100 fmol. But if
you really
get no bands at all  ( not just erratic band patterns),  something is wrong with
the template or primer or both ( I assume your kit does work on control DNA).

I hope this helps,

Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch

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