Nested Deletions

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Sat Aug 21 01:50:00 EST 1993


 wra at biochem.dental.upenn.edu (Bill Abrams) writes
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We are using an Exonuclease III/Mung Bean Nuclease kit to generate
unidirectional deletions of double stranded DNA. A difficulty that we have
encountered is a non-linear distribution of sizes after only short
incubation times at 28 deg C. Protocols designed to generate deletions of
approximately 250 to 300 bp would be most welcome. 
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Hi Bill,
    I have use this system to generate deletions of a gene I sequenced.  I
 did not use one of the kits but followed the method of Henikoff (1984) Gene
28:351-359 (Which is probably the same protocol).
 What I remembered was that I found all my short deletions (200 - 300 bp)
in the 3rd or 4th time point along with bigger deletions.  I found no 
deletions
in the first two time points. I screened using the size of a restriction 
digestion of minipreps, cutting  the plasmids on the other side of the 
restriction
site that was insensitive EXO III and once with one in the insert. This does 
mean 
you have to do a bit of restriction mapping of your insert but saves a lot of
time as screening with T tracks is too much work in my opinion.  I sequenced 
both
strands of a 3 kb gene in 250 bp chunks.  The small deletions were hard to 
find
but once I started looking in the later time points a found a few of them. 
Good Luck!
Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________




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