Dear Dr. Venkatesh,
Following are our conditions for the 3700 which may address the problems you are seeing:
We are encountering two kinds of problems.
1. Our sequences start with a good intensity in the beginning. After about
200bp the intensity suddenly drops.
I suspect the problem is your purification of the sequencing reactions, the isopropanol or ethanol precipitation method of purification of BDT sequencing reactions doesn't work well on the 3700 in our hands. We use the Millipore G50 96 well plate method to purify our reactions and resuspend in 30 ul of water. Since I don't have time to respond to many emails, please see Millipore's web site for a description of this method or contact your local Millipore rep for the protocol.
Note that for some reason which we don't understand it seems to still be necessary to dry down the reactions and then resuspend in 30 ul RNase free water. Merely eluting the reactions off the G50 plate with water and directly loading doesn't work as well in our hands. Also, one of the lots of DNA sequencing reaction standard which was sent to some of the early customers was not QCed by PE on the 3700 but only on the 377. This lot showed the same problem which you describe. As soon as we made our own sequencing reaction standard using PGEM and used the Millipore G50 cleanup this problem dissapeared for us.
The other parameters we have changed are as follows for the instrument setup and running to improve resolution and read length:
Run Temperature = 42C
Cuvet Temperature = 30C
Capillary Fill Time = 1800
Sheath Flow Volume = 17500
Run Time = 14000
Thanks to Steve Toth for the above suggestions. He is the best field application specialist at PE.
2. In couple of places we see a broad peak of G or T covering 3-5 bases.
This is also most likely your purification method. The broad peaks are dye blobs. For some reason the 3700 is more sensitive to dye blobs showing up. Using the Millipore method for cleanup will most likely eliminate the blobs.
Also I would like to know advantages of using water over formamide in
suspending sequencing reactions and also amount of water to use.
Water is free and doesn't degrade the sequencing ladders over time. However, it evaporates over time so it might be a good idea to the resuspend plates that will be run later in increasing volumes of water. We haven't determined the ideal volume to resuspend later plates in yet. However, the first plate run we resuspend the reactions in 30 ul.
Hope this helps. However, at this point I think all 3700 users are still learning and we would also appreciate it if other users would post tips. Particularly for Genescan runs since this has just been released and we are just starting to optimize this.
Best Regards,
Bob
_______________________________________________________________________
Robert B. Chadwick
Director, Genotyping/Sequencing Unit
Division of Human Cancer Genetics
Ohio State University
420 West 12th Avenue
Columbus, OH 43210
phone (614) 688-4783
FAX (614) 688-4761
e-mail chadwick-1 at medctr.osu.edu
web address http://gsu.med.ohio-state.edu/index.htm
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