DNA sequencing trouble - DNA sequencing (Sep/22/2006 )
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
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.
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.
That's the same we had, I recognize the name.