I sent in my prepped plasmids for DNA sequencing. Unfortunately, the results are very noisy (I attached a screenshot). I double-checked and the samples have the right concentration (100 ng/ul) and a good 260/280 ratio. I didn't send any primers because the company offers the ones that I need. Therefore, I am quite sure that the primers should work.
Maybe it is helpful to briefly describe my workflow; I did site directed mutagenesis via PCR and subsequent gel extraction. I then transformed competent bacteria and selected them. I used single clones for cultivation and performed plasmid preparation with a kit.
Does anybody have an idea what might be the problem? Are there any things that I am missing or pitfalls that can happen during my workflow and might not be obvious?
Many thanks in advance!
Can you attach one of the original .abi sequence files from the company please?
I have just run a test agarose gel of the plasmid isolations. Doesn't look pretty and the second half does not contain a marker, sorry. But still I think there might be an issue during the isolation. I would expect bands at the height of the one in lane 1. The brightness is caused by different concentrations, so that's okay. In the lower half, the bands should be at the exact same height. I know that plasmids can occur in different conformations, but the results in the lanes 10-19 still confuse me. Any suggestions? Thanks!
Regarding your sequence file and thank you for sending it.
The signal intensities are strong ( over 300 and many over 800 rfu)
The usual reason for this type of sequence is weak signal strength (less than 200) and high background from the capillary but this is not the case. This is 2 strong signals both of which are not affected by background noise.
The 2 common ways of getting this type of result are 1 A degraded sequencing primer missing 2 or 3 bases causing overlapping sequences.
and
2 the primer is good but is annealing in 2 places giving 2 completely different looking sequences.
There is no large unincorporated dye peak and strong signal strength over the whole sequence so it looks like the purification of the sequence reaction is excellent and the correct amount of both primer and template has been used.
As the primer is commercial my feeling is that it will be good quality.
You can see that wherever there is a run of the same bases (homopolymer) of 5 or more bases there are 2 or 3 in the middle of the run with no second base under the main signal.
My feeling is that the primer has annealed in 2 places (possibly eleven bases apart) giving you 2 sequences. Possibly in this case sequencing with a reverse primer might clarify the situation
The results of 13-19 are a surprise but i am not convinced that secondary structure effects are to blame for the size differences. How sure are you that this is a perfectly clean plasmid.? It has happened that researchers have borrowed plasmid from another colleague and the plasmid already has an insert or inserts. 15,17,and 18 run smaller thatn the expected size and I would have to blame secondary structure for this effect. Either sequencing or RE digest of some of the smaller bands and one correct size band might give some information as to insertion/deletion effects are possibilities
Thank you so much for your answers, Paul. I really appreciate the time that you must have taken for that! I resubmitted the samples using the respective reverse primers and will also do restriction digestion to test the plasmids. Again, many thanks! Have a great evening
- Hi, just wanted to give a short update. I tried another primer and it worked perfectly fine. Even though there were no multiple binding sites predicted for the other one, it might have been possible that it annealed to an additional site.
Well done Laura Charlier it is always a pleasure to get positive feedback on problems. I am glad that you sorted the problem
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