very bad signals after sequencing - bigdye 3100 (Jun/08/2010 )
dear everyone, I sequenced a plasmid DNA using AB 3130 sequencer and I got very bad signals and its not clear at all. I dont know what happened. can anybody give me some advice? my bigdye reaction is
plasmid: 2 ul (>200 ng)
bigdye (mixed with buffer): 2 ul
primer (1 mM): 4 ul
H2O: up to 10 ul
and PCR is
96C 1 min;
96C 10s,50C 5s,60C 4 min for 40 cycles.
and then I use ethanol/NaAc precipatation and resolve the DNA with formadine. and this protocol worked well before and I dont know why this time it didnot work.
It looks as if substantial amounts of labeled dNTPs are still present. I would be careful about the cleanup, perhaps using an additional 70% EtOH wash, or switch to a bead or column cleanup. You might also want to check the amount of template, which can be a problem with either too much or too little.
how was the signal strength on the raw data?
if it was weak then you have a lot of noise.
if it was strong then you may be looking at multiple sequences.
if multiple sequence then you either have more than one primer, more than one priming site or more than one species of plasmid.
Thank you for your replies.
I put the same sample on another sequencer, and this signal pattern never appear again. although I still didnt get 100% clear result, the data is better than that from the previous sequencer. seems like the sequencer didnt work well.
does your sequencer get maintainanced according to the suggested timeframe for regular check ups? Our 3130 just had a maintenance done a month ago and had better results than ever before for everyone using it. Before it was alright but sometimes you would get very low signals and even failed sequence analysis. There are also the daily and weekly maintenance which should be done religiously to ensure quality results