I have two PCR amplicon of 300+ and 400+ band size. but in sequencing it gave 562 (forward reaction ) and (242 bp in reverse reaction) for 300+ band. And there is no identity with forward reaction and complement of reverse reaction result.
Whereas for 400+ bp it gave 451bp in forward reaction and NNNNN in reverse reaction. What can be the reason?
Hi Munira,
unfortunately the electropherograms you have uploaded do not look good.
Ideally, you would want to see a single peak for each given position, while here you can see multiple peaks. For example, your file G09, at position 350 shows a green, a red and a blue peak. A clean electropherogram would show a single peak for each base.
The cause of this might be:
- you have multiple copies of your gene and your primers are amplifying them.
- your primers are not selective enough (therefore they match multiple sections of your sequence.
- you amplified something different. Maybe you were aiming for plant DNA or insect DNA and you amplified bacterial DNA instead (or in addition).
Unfortunately, you will have to try to re-amplify.
Maybe try and check if your primers can be improved, too (are the primers very generic? if so, that might be an issue!).
Good luck!
I used cDNA from fish RNA, my primers are degenerate but they picked my expected gene when tried in blast. and yes there are more than three gene in the same family.
Hi,
As mentioned above by Dr. Francesco Martoni, the sequence profiles cannot be used for deducing any sequence. There is too much mixing. If you are directly using an amplified cDNA fragment to sequence, it will be difficult particularly as you mentioned there are multiple genes in the family. Better to clone the fragment and sequence using plasmid specific primers. It would work
Thank you so much for your informative suggestion. I will try to improve primer sequence and will go for cloning.
Did you get a single clean amplified pcr band to sequence?
What temperature did you anneal the primers to get the pcr product?
What was the annealing temperature of the primer for the sequencing reaction and is it the same primer that you used to do the pcr? What are the pcr primer sequences?
To me the problem looks like the sequencing reaction is not working at all. The peak intensities are very low( some are 30-40 and should be 600 or more) so the background noise is nearly as strong as your sequencing signal.. On some of the samples the unincorporated dye peak is very strong which mostly happens when the sequencing has failed and the purification of the sequence reaction cannot remove all of the dye peak.
Is anyone else getting good sequencing results with this kit for any other amplimer?
yes the bands were sharp and single,
I used 57 and 58 for two different annealing but what annealing temperature used for sequencing I do not know. In case of others , they got reliable result from that organization.
Thank you Munira,
Sequencing annealing is usually done at 55 so as your primers work at 57 the problem is not that the primers cannot anneal in the sequencing reaction which I thought may be possible as the primers are degenerate. As this is commercial sequencing we can assume that the sequencing reagents are top quality. Since the very low intensity of the electropherogram peaks indicates that the sequencing reaction has not worked then the 2 most likely causes of sequencing failure are 1....not enough dna template
or 2 not enough sequencing primer was used in the sequencing reaction
this is a bit strange, but please re-sequence the sample. Once I had the same problem. the result from the sequencing was not the same with the gene that I amplified. Then we realized that sequencing companies sometimes mix the sample codes, so you receive someone else`s sequencing result.
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