exoIII is a dinosaur.try dnase1 or tn1
suter at de.d400.mpg.MPIZ-KOELN.VAX
suter at de.d400.mpg.MPIZ-KOELN.VAX
Wed Sep 22 06:48:17 EST 1993
as reported by: S=rander;OU=cco;O=caltech;P=edu;A=d400;C=de
with a question by me at the bottom:
+TRANSPOSON FACILITATED DNA SEQUENCING
+PNAS vol 88 pp1247-1250 feb. 1991. The materials are now available from
+GOLD Bio Technology (800) 248-7609, in Mo. (314) 647-2509.
+I do not work for them and I will not supply the materials because they have
+been upgraded and because GOLD has the license. I don't know the cost either.
+This method is based on the random insertion of a transposon. Each resulting
+colony has the transposon in a different place. The vector is constructed
+to select against insertions outside of the "insert". This works 95% of
+the time. To determine the insert location PCR primers are made to sites
+in the vector polylinker and to the inverted repeat at the ends of the
+transposon. There are unique sites on each end of the transposon for use as
+sequencing primers later on. Using PCR amplification with one end primer
+and the IR primer and a second reaction fron the other vector primer and
+the IR you can determine the distance to the insertion site. Then by
+selecting the apropriate set of colonies you can sequnce the whole thing
+in both directions. I did a 6.5 Kb clone in 3 weeks from start of mating
+to finished Sanger sequencing. Reading took longer but that's another
+There are some probblems encountered with this method. One you have to have
+threee strains. This is trivial but adds two days over what I will describe
+next. The only other trouble is that occasionally we got expression of
+the insert which was toxic to the bugs. We know this because we removed
+the first part of the coding region and things were fine. I like this
+method alot and I have used it for 2 years. I will never do an EXO III
+deletion agaain to generate sequencing sets.
+GENERATION OF BIDIRECTIONAL DELETIONS USING DNASE 1
+Biotechniques Vol 14 No. 3 1993 pg 408 -411
+DNAse 1 cuts both strands in the presence of Mn2+. By allowing random cuts
+on a frequency of ~1 cut per insert molecule and having different ends
+cut to leave a 5' overhang two sets of nested delteions are generated.
+The paper is very clear and so I will not go into all of the details.
+Suffice to say that no special strains are needed and not matings have to be
+performed. What you get is a nested set which is mapped in a similar way to
+the transposon method but you only need polylinker primers.
+Drawback: you have to isolate the insert.
+Variant: use 4 hitters which leave blunt ends.
+This method was submitted by James Eberle from the Dept. of Bio Syracuse
+University. A tip of my hat to him and/or those who helped design this
+method. I think it is the fastes most straightforward of the methods we
+have tried. You also end up with some potentially useful deletion clones.
+I would ask that any questions be held until you have read the papers or
+contacted GOLD Biotech. I posted this information because I spent months
+screening EXOIII sets before I could sequence and I know what a pain
+that can be.
+Best of Luck to All
Big and obvious question: how do i contact manufacturers of these systems
here in germany ?
Dr. Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10, D-50829 Koeln, Germany
Tel.xx49.221.5062-221 Fax.-213 e-mail: suter at vax.mpiz-koeln.mpg.d400.de
if this is victory, our hands are to small to hold it
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