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DNA sequencing trouble - DNA sequencing (Sep/22/2006 )

Dear All,
please help me out with the problem i am facing right now with my dna sequencing,
i have been doing DNA sequencing for a couple of months, i am now working on sequencing the entire genome of a virus.
for the past one month i am facing some problem with the sequencing and i am unable to figure out what is wrong in the procedure, my sequencing reactions and the ethanol precipitation steps and protocols have worked before so i cannot doubt them, i checked my DNA sample via gel electrophoresis before putting for sequencing reaction, the primers have worked very well before, i even tried to change the 5X buffer and the Big Dye but nothing helped, so i was doubtful about the machine, so i reran the samples that had given good results last month and this time i got good results, so the machine is working well,
so my next target was the sample i was using, meanwhile other people in the department also ran their samples and they too didnot get good results, i am lost completly and cannot figure out whats the problem, can any one of u help me out, i am totally stuck up here and unable to proceed further in my research.
Thanks in advance
paapu

-paapu-

We also had problems one time in the lab. There was something wrong with the needle that pipets it in the tube (they told me, I don't know much about the machines).
The problem was solved when we started to use double the amount of the solution you resolve your pellet in (is that the big dye?). I hope this can help you!
Otherwise, maybe contact the company from where the machine is.

-aspergillie-

I'd like to look at an electropherogram of your sequencing results. What machine are you using? I would check the capillary current during the run -- I believe it is automaticaly recorded. As aspergillie reports, the injection requires both the capillary and the platinum needle to be submerged in the sample, so a height adjustment of the plate can make a big difference.

-phage434-

QUOTE (phage434 @ Sep 23 2006, 10:22 AM)
I'd like to look at an electropherogram of your sequencing results. What machine are you using? I would check the capillary current during the run -- I believe it is automaticaly recorded. As aspergillie reports, the injection requires both the capillary and the platinum needle to be submerged in the sample, so a height adjustment of the plate can make a big difference.


Hai Phage 434 and Aspergillie,
thank u both for ur suggestions, i shall look into the capillary and platinum needle, by the way it is an automated sequencer from ABI prisms.
-Paapu

-paapu-

That's the same we had, I recognize the name.

-aspergillie-