The fluorescent labelled primer for the expected sample is purchased 2 years ago and for the sample with intense signals is purchased 3.5 years ago and have been kept in the identical condition ever since.
I am not sure which is the problem ....the huge small dye peak which has been cut off by the analysis software or the fact that your real amplimer has changed from dimorphic to monomorphic. The large dye peak at small size could be primer dimer or possibly if the primers were stored dissolved in water (or exposed to light a lot) then they are not protected from nucleases and a lot of your primer could be degraded. You still get a strong signal from the amplimer and you can start the analysis after the small peaks to get better results.
Hopefully the 2 pictures are from different dna samples but if they are from the same sample then it may be that there is a polymorphism under the 3' end of one primer and you are only amplifying one chromosome so it might be worth lowering the annealing temperature 3-5c to see if both alleles show
Hi Paul,
They are different samples and loci. One forward primer purchased 3.5 years ago (with high small peaks) and the other (good small peaks) 2 years ago. The reason I used electropherogram of different loci for this question is because although I don’t have access to the older primer’s very first electropherogram, they looked very similar to the good small peaks of this different loci. Both primers are around 20bp but if you see the older primer have huge peaks that are smaller than 20bp whereas the newer primer have the hugest peak at around 20bp and others aren’t as huge.
Good that the samples are different. The primer peak has to be enormous because pcr relies on the high concentartion of primer allowing the primer to anneal to the single stranded template before the 2 strands of template re anneal so the peak height is normal. Often analysis software ignores all peaks leass than a fixed percentage of the first peaks so to avoid non reading of size ladder peaks the analysis is usually started after the unincorporated primer peak otherwise most peaks would be ignored.
I think that the peak lower than 20 bp is made up of degraded primer due to primers being made up in water ( which is quite acidic to single stranded dna). This is less likely to happen with primers stored and dissolved in TE as the Tris buffers alkaline and the edta removes divalent ions and stops nucleases from being active. You also have to accept that the resolving power of linear acrylamide is really not very good at 15-20 bases but I do think that your smaller peak effect is real but the good peak size of your amplimer suggests that the level of degradation is not high enough to spoil the pcr yet due to the very large excess of primer initially in the reaction
Does the following part of your answer mean that the ladder could bleed into the sample analysis? “Often analysis software ignores all peaks leass than a fixed percentage of the first peaks so to avoid non reading of size ladder peaks the analysis is usually started after the unincorporated primer peak otherwise most peaks would be ignored.”
Because I have got half of my samples from the plate offscale too and do not know why this was the case - in all of them there are huge small peaks?
The large signal from very small dna is to be expected since the sample loading into the capillary is electrokinetic so loading is more efficient with small (very mobile) and charged molecules so the sequencer loads small highly charged fragments preferentially. If you loaded the plate again you would expect better results because you have desalted the input sample to some extent during the first loading.
The answer may be to re analyse the plate results starting a few minutes later so as to exclude the huge salt peaks (primer peaks). Speak to whoever runs your samples /tech help to discuss re analysis.
What I meant by my earlier answer was that to avoid too many peaks being analysed which are just background noise there is a fixed percentage cut off defined by early peak height under which peaks will not be analysed. As the ladder is often quite a weak signal when there is a large early peak the software may ignore the ladder peaks making the sizing part of the analysis impossible since some or all ladder peaks are being ignored. I used to run my samples at 2 dilutions to try to get round this problem. Again re analysis with inclusion of more peaks by tweaking the analysis parameters should help
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