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:
modified parameters: injection voltage: 1500V
injection time: 20s
foil piercing: ON
flat field corrected: off
tank levels: OFF
The rest are the default parameters:
Default parameters: run temp: 50'C
cuvette temp: 40'C
cap pressure: 1200psi
cap fill time: 1200s
max current: 800uA
pre-run voltage: 6000V
Pre-run time: 300s
sample volume: 2.5ul
wash volume: 20ul
run voltage: 5250V
sheath flow vol: 12000 counts
sheath flow period: 942msec
data delay time: 1500sec
run time: 10000sec
These conditions did away with the "front-loading" problem and we are
now getting reads averaging 840 bases with a phred Q>20 of around 500
(+-50) using dsDNA templates and BigDye terminator half reactions (we
think we could go down to quarter reactions without losing quality and
will be trying that in the future).
We were lucky enough to get one of the foil piercing units and that has
saved us a lot of problems. Without it, we had to increase the volume
of each plate by 15 microliters to take into account evaporation (25ul,
40 ul, 55 ul, 70 ul). The foil piercing unit removes the evaporation
problem and I suggest getting it if you don't already have one.
> Also I would like to know advantages of using water over formamide in
> suspending sequencing reactions and also amount of water to use.
We found no difference in quality between using formamide and using DI
water, so we just use water. Saves the expense and time of making DI
> 2. In couple of places we see a broad peak of G or T covering 3-5 bases.
> Initially we thought problem maybe due to formamide. Changing formamide did
> not help. We considered problems with purification. We are using 56%
> isopropanol. Increasing or decreasing isopropanol concentration did not make
> any difference. We also tried spin columns and problem still persists.
> I would appreciate input from experienced users.
We also had this using just a 65% isopropanol (final ) ppt. Getting
rid of the excess unincorporated labeled dNTP's using an additional 70%
ethanol wash solved the problem for us. Using spin columns (or plates
with g50 in them) should also solve the problem, not sure why it did not
for you. Try the 70% wash and see if it helps.
Stephen R. Lasky, Ph.D. #
University of Washington #
Department of Molecular Biotechnology #
srlasky at u.washington.edu #