Dear all,
In the past 6 weeks we have been experiencing problems with the read
length of our sequences. Since it was only 1 lab that seemd to be
getting this problem, we tested new primers and concentrations, new
sequencing kits (Amersham) from 2 different batch numbers, new DNA
Miniprep kits, (DNA quality and quantity ascertained on an agarose gel),
PCR machines and conditions were changed and a number of other variables
- nothing really seemed to solve the short read length (sometimes as
short as 100 bases, sometimes as long as 350-400 bases, with the
sequences dying out rapidly or becoming very messy). Signal strength
was far too low on many samples (frequently below 100, often below 50).
Most other labs contributing samples to be run on the ABI 377 were using
ABI kits, or Amersham kits dating from before July, and had no read
length problems.
Due to the nature of some of our samples (those containing
microsatellite repeats) some will always be short read - but generally
never so many so consistently, and even when they don't contain a
repeat, they are still too short. When we tried ABI's new Rhodamine
kit, the results were up to 450-600 bases with the same samples.
Then we changed back from the Amersham kit to the 'old' ABI sequencing
kit last night, using all the original parameters - all the sequences
were long read (no N's until 400-600 bases, for more than 90 per cent of
the samples.) Signal strength generally between 150 - 300.
Has anyone else had this problem with the Amersham kit? This problem
can be traced back to late July - a bit before the time that Amersham
had to call back several batches due to migrating A's and an interfering
high T background. Before this occured, we had been using the Amersham
kit very happily (especially at their more competitive price!) for
several months with no problems at all.
Amersham has been contacted, so we hope this will soon be solved!
Chloe Aldam
Long Ashton Research Station
Bristol
UK
