lyok, on 18 June 2012 - 11:38 AM, said:
I am not really sure what you mean with cleaning.
By cleaning, you simple mean cleaning the sample, removing components, like salts, that you dont want?
I find it hard to understand that the first bases would be bad and after a few bases it would become better? The "dirt" in the sample isnt removed, so how come it gets better all of a sudden?
I am not sure what you mean with this: as with any electrophoresis method, you get close early peaks and diffuse late peaks. How come you get early peaks?
And next gen sequencing? I dont sequence the samples myself, we ship them to a company , so I would find it weird they arent using next gen sequencing.
(by next gen sequencing, you do mean illumina etc?)
yes (regarding next gen sequencing). next gen is not sanger sequencing.
i work with a beckman-coulter ceq 8000. it works by separating sanger (dideoxy termination) sequencing samples by capillary electrophoresis (similar to abi sequencers). the early bases come through tightly packed and usually saturated (fluorescence), making it difficult to properly position the bases. the late bases come through as broader, more diffuse, peaks so you may get false base calls.
as for cleaning, after running the reactions, you clean up the sample and suspend the fluorescent dna fragments in formamide (water for abi, i think) for electrokinetic injection onto the capillaries.