I'd like to share our experiences from using the ABI 310 for DNA
Sequencing. If there is anyone else out there who uses the ABI 310
for sequencing and has different/additional experience, I would be
very interested to hear your story.
We've received the ABI 310 about a year ago. First of all,
this instrument must have a service contract, since the laser life
span is no more than 1 year of normal (4-5 days/week) work. We
already have had a couple of service calls (new laser installation),
and I have to say that the service was excellent.
Our first experience with the 310 was relatively disappointing. See
the data below:
---------------------------------------------------------
Seq. kit Avg. # of bp Avg. % Accuracy
ss DNA
----------------------------------------------------------
Big Dye 506 93.3
Amersham V.1 496 96.6
Amershan v.2 428 90.7
---------------------------------------------------------
---------------------------------------------------------
Seq. kit Avg. # of bp Avg. % Accuracy
ds DNA
----------------------------------------------------------
Big Dye 470 91.4
Am. v1 402 95.3
Am. v.2 443 94.6
dRhod 496 90.3
----------------------------------------------------------
DNA used: M13, pGEM, pUC19
Capilary used POP6
All data unedited
Conclusions:
1. More bases sequenced with ssDNA than with dsDNA.
2. Amersham v.1 - most long & accurate reads.
After these experiments we've contacted techsupport at ABI and
Amersham. ABI's response was simple - follow our guidelines.
Amersham was more responsive, and following are their
recommendations for possible solutions to get better runs:
- with ds DNA, increase beginning denaturing to 4-5 min
- decrease DNA concentration (400 ng for plasmid)
- use 12 mkl TSR
- optimize annealing temperature
---------------------------------------------------------
DNA Length % Accuracy DNA, mkg TSR mkl
of read
----------------------------------------------------------
M13 691 97 0.25 12
pGEM 706 95.8 0.4 12
----------------------------------------------------------
5 min denaturing
Amersham v.1
All data unedited
One can see considerable increase in average length of read while
maintaining the same % of accuracy.
Important note: All credit for experimental work should go to my
associate Bonnie Liscek.
If there are more questions we'll be glad to answer them.
Thanks,
ma
M. Alterman, Ph.D.
Director,
Biochemical Research Service Laboratory
University of Kansas
Lawrence, KS 66045
Phone: (913)-864-4166
(913)-864-4247 lab
FAX: (913)-864-5396
http://www.idl.ukans.edu/anylresc/brsl/brsl.html