Stephen R. Lasky <srlasky at u.washington.edu> wrote in message
news:7qg5er$q1p at net.bio.net...
> I responded to this message once before, but had some trouble getting it
> to the newsgroup. Lets see if it makes it this time, if it doesn't make
> it, I will assume I still have the problem:
>>Tvenkatesh at synapticcorp.com wrote:
> >
> > 1. Our sequences start with a good intensity in the beginning. After
about
> > 200bp the intensity suddenly drops.
>>> Is the intesity of the first few bases really high? We found that a
> situation that we called "front-loading" caused the first couple of
> hundred bases to have a very large signal (maxed out on the trace), and
> then the signal would die out rapidly. We found a solution to this by
> modifying the loading conditions:
[other loading conditions]
Hi,
we are using the ABI 310 with the big dye terminator chemistry. We ,
recently , had the same problem in a lot of runs of the same batch : a very
high signal that died out after 100 bases. We looked back in the records of
older exp. and found a number of samples with the same characteristics. On
agarose gel there should be inserts ( in pUC18) of 600 bp where we could
sequence only the first 100. When we analysed all the samples , that could
still be read with some accuracy , with software for detecting loops (gcg
stems and loops) We found that all of them had strong internal structure.
So
5'-ACGTTGCCGAATTGGGTTAAGCTTA--------------
TTAACCCAATTCGAAT-------------
is sequenced till base number 10.
In our case just by change. We hypothised that the structure prevented the
sequence reaction to proceed. We changed the cycle sequencing parameters for
the reactions (higher extension temp ; see the ABI web site) which solved
the problem in our case.
--
Gys
