Hello,
I sent 1 plasmid with 3 primers for Sanger sequencing last week, but all reactions were of poor quality. As the troubleshooting suggests to almost every problem to send PCR fragment instead, I amplified 2 of the regions. However, only 1 fragment amplified, when I was going to purify it, so I purified this 240 bp fragment and sent it for sequencing with both forward and reverse primers (because I was wondering if it could be some unspecific amplification).
Of these, only the forward primer worked, however, some of the peaks were wide, so it was read as 2 nucleotides (see attached picture). What could be the reason for this? Especially at the beginning of the chromatogram (it was only up to 100 bp or so)?
What could be the reason, that one of the sequencing reactions didn't work, if the other did and it confirmed that there is the primer annealing site?
What would you suggest next? I can amplify the insert with two sets of primers (each yielding 4-5 kb amplicon) and send them for sequencing again or isolate the plasmid again and send it for sequencing. Which option would you recommend?
Thank you
Sequencing is always poor for the first 30 bases after the end of the sequencing primer so to get theese bases it is necessary to run reverse primer sequencing. I suspect the amount of sequencing primer or template dna is to blame but can you attach a typical sequencing file ( *.abd) file as supplied with your sequence by the sequencing facility please
Is your plasmid region of interest flanked by any commonly used primers (e.g. M13)? It's a feature built-in with the multi-cloning site for many commercially available plasmids.
If yes, use those primers for both amplification and then use them for sequencing.
Good luck!
Hi Paul Rutland
thank you for your answer. I know that the first ~30 nucleotides cannot be read, but that's not what I meant. I attached the original *.ab1 files to the question above. There is also picture with arrows to show what I mean. It's already in the sequence, where the peaks are nice (in the region ~50-110 nt) and the peaks are just slightly broader than the other peaks and thus it is interpreted as two nucleotides.
The one with broad peaks ends with 15; the one ending with 16 is read with reverse primer and is quite messy (although it looks like good peaks with high background - out of curiousity, I now tried to "read" it manually and at least the end of chromatogram correspondences to my sequence).
Hi Katie A S Burnette
thank you for your suggestion. Unfortunately, it's home-designed plasmid :-/
There is GFP on one side, but neither of the primers that I found anneals to that sequence :-/
On the other side, there is the tetracycline resistance gene, for which I found 2 primers, so I guess I could get one of them.
However, that shouldn't be the problem. We have primers for outside of the insert, those were two of the original sequencing primers that I used in the first round.
So I take it that you recommend to PCR amplify and sequence?
hello Tomáš Hluska
thank you for the files which show that the reagents used are in date and the signal strength greater than 3000 (300 is the lower limit for ok sequencing). Sample spread can be caused by poor loading of the linear acrylamide matrix and there are a few odd extra bases under some real bases which might suggest poor capillary filling but I think that it is more likely that the early multiple bases is a mixture of poor basecalling algorithms and the constitution of the sequenced sample.
So the KB basecaller can be inaccurate at early base discrimination and often the odd bases can be resolved by re analysing with an "even spacing" type basecaller. This works when you have your own sequencer but I do not know if Genewiz offer this service when data is poor.
Very likely is that the sample loaded on the capillary is contaminated with small salts as can happen when mini columns are used to purify the dna (plasmid). This causes mobility issues on the capillary and the solution can be to add EDTA to the sequence reaction and re purify it before running it again and again some sequencing companies will do this when the spread is severe
Troubles with plasmid Sanger sequencing can arise from several factors, including: 1. **Inadequate Template Concentration**: Insufficient concentration of the plasmid DNA can lead to poor sequencing results . 2. **Impure Template DNA**: Contaminants such as salts (EDTA, NaCl, NaAc, Kac, KCl), proteins, detergents (SDS, Triton X-100), RNA, chromosomal DNA, and organic chemicals can interfere with the sequencing process . 3. **Anionic Contaminants**: Anionic contaminants from "kit-purified" plasmids can affect the sequencing reaction . 4. **Excess Salts**: High concentrations of salts can disrupt the sequencing process . 5. **Poor Quality or Inadequate Primers**: Incorrect or low-quality primers can lead to failed sequencing reactions . 6. **Absent, Deleted, or Mutated Primer Binding Sites**: If the primer binding sites are absent, deleted, or mutated, the sequencing reaction will fail . 7. **Air Bubble or Block in the Capillary**: Physical obstructions in the capillary can prevent the sequencing reaction from proceeding properly . 8. **Secondary Structures in the Template**: Structures like hairpins or long stretches of Gs or Cs can cause the sequencing polymerase to stop prematurely . These factors can individually or collectively contribute to the failure of plasmid Sanger sequencing, necessitating careful optimization and troubleshooting of the sequencing conditions.
Dea Paul Rutland
so it's likely problem on their site? If I should run the sequencing again, it should be OK hopefully?
If you run it again I would 1 purify less plasmid and
2 give the bound plasmid a second wash with wash buffer before elution to minimise the carry over of small salts from the purification process and expect better results early in the sequence
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