Exonuclease deletion kits...help !!!

David Micklem drm21 at mole.bio.cam.ac.uk
Thu Apr 18 15:13:30 EST 1996


In article <Pine.SUN.3.91.960417161109.27015C-100000 at bio02>, Sensational
Gravity Boy <ed at bio02> wrote:

>Hi all,
>
>We're looking into purchasing kits for the exonuclease deletion of clones
>to generate sequencing templates (for example Erase-a-base from Promega). 
>We'd love to hear about kits which have proved reliable, user friendly, etc.
>I know lots of you out there use such kits, so please, please, let me 
>hear from you.
>
>thanks,
>
>Ed Taboada
>Dept. of Biology
>University of Ottawa, Canada

[Ed, your return Email address is incomplete, so I am posting this]

Hi Ed,

I know that you are asking for ExoIII type kits, but could I put in a vote
for an alternative system that has worked superbly well for me, and others,
here.

The method is described in Strathmann, M. et al PNAS Vol88, 1247-1250,
1991, and involves the use of bacteria carrying the gamma-delta
transposon.  Its very quick and easy, with bugs doing all the hard work! 
Basically, you transform your plasmid into a donor strain (F+, KanS,  
RecA+, I think).  These cells are mated to F-, KanR, RecA- cells.  At low
frequency, the transposon, present on the wildtype F, inserts at a random
position in your clone.  In the donor cells this forms an unresolvable
co-integrate, which is transferred to the recipient cells during
conjugation.  Inside the recipient, the cointegrate resolves into
(plasmid+transposon inserted randomly) plus F factor.  You plate the
conjugation mixture onto KanAmp plates, and the only things that survive
carry your plasmid with an insertion.
Insertions can be mapped by restriction enzyme or PCR. Due to unique
subterminal sequences on the transposon, each insertion allows sequencing
in both directions from the insertion site. This really helps in getting
complete ds coverage.

I got appropriate bacterial strains as part of a kit ("Tn1000") from Gold
Biotechnology. (I have no connection with Gold Biotechnology except as a
customer).  The kit also contains a minimal vector, based on Bluescript. 
Using this cuts down the background of insertions into the vector instead
of the insert, but is not essential.  In general the instructions and
primer sequences described in the paper, especially for mapping inserts,
are much better than the kit directions.

After struggling to get a good deletion set with exoIII (Erase-a-base
actually) for one gene I sequenced, it was a joy to start using this
system.  I can't recommend it highly enough!

I hope this helps.  Feel free to ask questions if the above is unclear.

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)1223 334089               
_____________________________________________________________



More information about the Methods mailing list