sequencing problems: chromatogram attached - messy peak data (Jul/18/2008 )

Hi everyone,

I am currently trying to set up an ABI 3730 Sequencer and I have been getting choppy uncallable peaks. I have attached a picture of the chromatogram. The DNA is a plasmid that has been preped. The concentration is 150ng/ul via nanodrop and have a 280/260 ~1.8 so I believe it is pretty clean. Running the plasmid after RE digest on a gel shows a single plasmid band with a single cut insert band.

Cycle sequence parameters with M13f/r primers are

96C 5min
96C 10sec
50C 5sec
604min
25cycle
4C o/n

I cleanup using millipore dye cleanup plates. I run them on a 50cm array along with the BD std seq. The std comes out perfectly fine but only 2 out of 50 produce viable results. The rest of them produce sequences like the attached chromatogram. The only thing I can think of is the millipore cleanup plates that may be messing it up. Does anyone have any suggestions? Thanks in advance!

How much DNA are you adding to your reaction?
Clare

This looks like what you get when either you have no DNA, or when your primer fails to bind to the DNA you have. Or, no primer. The dye blobs are pretty clear indication that the instrument is working pretty well.

After nanodropping the plasmids, I am adding around 300ng of plasmid dna, plasmid size is about 3.5kb. This should be enough DNA. Just throwing this out there, would minor protein contamination cause any problems? The primers are M13 F (-40)/R which are universally used primers so they should work. I am using 50C as the annealing temperature. Primer concentration is 4pmole per reaction. Thanks for everyone's input! Anymore suggestions as to why this might occur. I might try increasing both the primer and dna concentration. From the nanodrop 280/260, it seems that the dna is not contaminated but I am afraid that if it is, if i were to add more DNA, more contamination (whatever it may be) may be added into the reaction inhibiting Bigdye too. Any suggestions? I am currently using a home made plasmid purification procedure with glass filters to bind the DNA and eluting in pure h20.

The M13 primers are common but by no means universal. Are you sure your plasmid has those primer sites?

Yeah, the plasmid does have M13 sites. In the experiment, I would choose about 24 clones and plasmid prep all of them and sequence M13 F and R (separate reactions of course =) ) in 48 rxns. I would get 4 rxns that provide good data and 44 rxns that would produce chromatograms like the one I attached in my first post. Which is weird because those 4 rxns would act like the positive control (sequencer works, big dye works, cleanup works)...so is it the plasmid quality? but the rxns would be weird, a forward rxn that worked for one clone would have a reverse rxn that did not work or vice versa....it is weird results which stumped me for now...

Helo everyone...does anyone have any other suggestions....i still haven't been able to trouble shoot this problem.....thanks in advance!