Dear colleagues, we are a group of microbiologist’s at the University of
Southern Denmark, who are starting up a sequencing facility. At the
moment we have access to an ALF and an ALFexpress sequencer. Both
sequencers are equipped with version 1.0 of the ALFwin sequencing
analysis software. We have problems to obtain a good DNA sequence
quality.
The documentation that follows the Alf sequencers are very poor, and the
trouble shooting guide on Pharmacias homepage can not answer my
questions. My questions are:
1) Is there a upper limit (or optimal value) of the power settings
during a run?
2) Is there an optimal ionic strength of the running buffer (i. e. conc.
of TBE) ?
3) Is there an optimal ionic strength in the gel (i. e. conc. of TBE) ?
4) And how does the ionic strength and power value effect the resolution
in the sequence trace ?
5) How do you treat the samples before loading on the gel ?
Log values from our samples compared to a demo1.alf file obtained from
EMBL's software archive /gs/demo_dta directory
Our ALF: exp. cond: 1500V 60mA 25W 55C 3mW
19) 655V 28mA 25W 45C 3mW
875) 1261V 19mA 24W 45C 3mW
Our ALFexpress: exp. cond: 1500V 60mA 25W 55C 0mW
19) 352V 60mA 21W 55C 0mW
918) 620V 40mA 24W 54C 0mW
EMBL's Demo1.alf: exp. cond: 2000V 60mA 60W 50C 4mW
4) 1781V 31mA 56W 50C 4mW
808) 2000V 20mA 41W 50C 4mW
The sequence trace from ALFexpress looks pretty awful, the trace from
ALF much better but not even close to the quality of the trace from
EMBL's demo.alf file.
The technician who prepared the gel of the ALFexpress used a recipe that
gives a final TBE concentration of 1.5 in the gel and 0.6 in the running
buffer. The gel was prepared from a Plus One ready solution from
Pharmacia. The ionic strength during the Alf run is unknown but the gel
and buffers were prepared according to the suggestion in the manual from
FMC. The gel was prepared from a LongRanger gel solution.
I hope you can find the time to send a short answer to this email
yours sincerely
M.Sc. Jesper Andersen (Ph.D. student)
