Cocea cocea at
Wed May 31 05:52:06 EST 1995

Dr I.M. Clark (imc1001 at wrote:
: I have been attempting to make deletions from an 850bp fragment in pBLCAT3 
: using Promega's Erase-a-Base.  Screening the final colonies yields almost 
: entirely 'random' sized plasmids, and I lose my insert entirely.  This is 
: despite the fact that the deleted plasmid prior to re-ligation appears to
: be sequentially deleted across my time points.  Any tips, hints or just
: comments would be appreciated.

: Ian.

: --
: Ian M. Clark
: Rheumatology Research Unit,
: Addenbrooke's Hospital,
: Cambridge. UK.

Hi- I had the same problem when I tried to sequence a 2 kbp DNA fgr. using
the same kit. The "nested deletions" seemed ok, i.e. their sized decreased
as expected. I have sequenced in both directions and I have finaly managed
to read about 600 bp located near "the 5' end", but never got the 3' end.
We have blamed it on the polymerase's incapacity to resolve difficult 
secondary structures, but who knows? Given the fact that many sequencing
reactions gave blank lanes, I thing Exo III eats up the sequence for the
annealing primer, and thus there was no initiation. What I thought to do next
was to add a stuffer to protect this end while digestionthe other one with
Exo III (see Maniatis) - then remove it before ligation, but then I had to
forget about the whole experience and I still don't have a clear explanation.
Maybe you'll be luckier!

Regards, Laurentiu COCEA (cocea at

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