We use an AB 3130xl Genetic Analyzer for Sanger Sequencing, and on occasion, we see our plasmid samples struggle with wavy, loss of resolution (as seen in the attached image). Our general assumption is that a contaminate is co-migrating during electrophoresis, but I'd like to know if anyone has narrowed it down to more specifics of what those contaminates might be?
I'd like to provide better troubleshooting suggestions to people on what is causing this issue. Any advice/suggestions could be greatly appreciated! Thank you.
It would be good to see a .abi sequence file not a picture as there is a lot of diagnostic information in .abi files. The trace does have characteristics of template overload or primer dimer so there is a strong initial signal falling away rapidly as the sequencing reagents and primer get used up but without the original file it is not possible to see the exact extent of the signal strength
I sometimes got this kind of messy pattern with plenty of overlapping peaks when the primer failed to hybiridize to the template, i.e. when the sequence complementary to the primer was absent. How does the height of the peaks compare with that obtained with a clean chromatogram yielding a good sequence ?
Firstly l would suggest to check the plasmid quality specifically the 260/280 ratio and also check on gel. If the quality is poor redo the plasmid extraction and ensure there is a clean up step before giving sample for sequencing.
Hi Everyone - thank you for the responses! We have been troubleshooting some reagents, and we suspect our Dye has gone bad; we are setting up a test tomorrow using a new tube of BigDyeTerminator to see if we can get any clarity. If so, I'll follow-up with you all on our results!
Paul Rutland I can't find the exact .ab1 for the screenshot I provided, but I'm attaching the .ab1 of another example of the LOR we've seen on plasmid samples.
Pierre Béguin we've seen this in our pGEM control sample as well, with the established plasmid/primer combo (M13 primer). The RFU/peak heights are sometimes comparable with "good sequences" and then sometimes it's on the low side.
Vanita Malekar at first I was thinking it was an issues with the samples, however, our control pGEM plasmid is also showing the same LOR. Ultimately, I think this same LOR pattern can be seen from a wide range of issues, but since it's in our controls, and we have noticed it getting worse, we think a reagent has gone bad.
Thanks everyone! I'll keep your suggestions in mind while we continue to troubleshoot the issue!
Thanks for the file Lisa McDougall . I think that you are right about it being the sequencing mix. Even though this is a re injection( loads more dna onto the capillary as the first run has further desalted the sample) the peak heights are only about 200 and the expected for a 3130 are about 1000. The trace starts at the right place so salts in the sample are not to blame. This would cause low peaks but the trace would start later on as the salts loaded first. One way of achieving this type of result is to carefully thaw the seq mix. A lump of pure ice floats on top and then if you pipette out the right volume of seq reagent from the top of the tube it is mainly water. Mixing of the reagent mix is essential.All other aspects of the run ( current,temperature,matrix age etc look good) I would be interested in the result of your reagent test.
We have some clarity!
Of course it was the last thing we checked! Our Sanger reaction cleanup method was a bad lot, and was causing the problem. The company is troubleshooting the lot-specific problem, and has since sent us a replacement lot of reagent and our issue has resolved. We have consistent, beautiful reads in our control plasmid samples again! Thank you all for the suggestions and input!
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