I amplified my PCR fragment from mouse genome, and it gave 1 band. However, when I sent it for sequencing, it gave double signal in some regions. So I would like to ask if anyone have experience how to sequencing this PCR mixture with same size. Thank you so much.
1) Use an internal sequencing primer that primes only one of the two fragments.
2) If you have sequence information for your band, try digesting the DNA with restriction endonucleases that don't cut your band but cut down the other one far enough away to allow gel purification of your band.
3) Clone the mix into a plasmid vector (blunt or TA), pick clones, and sequence the insert of enough clones until you get the desired number of sequences from your band of interest.
4) Research further for a method much more high tech than mine above!
Good luck!
1) Use an internal sequencing primer that primes only one of the two fragments.
2) If you have sequence information for your band, try digesting the DNA with restriction endonucleases that don't cut your band but cut down the other one far enough away to allow gel purification of your band.
3) Clone the mix into a plasmid vector (blunt or TA), pick clones, and sequence the insert of enough clones until you get the desired number of sequences from your band of interest.
4) Research further for a method much more high tech than mine above!
Good luck!
Hi Christopher, thank you so much for your very kind reply. Since it is transgenic mice, and we still do not know the sequencing information due to the unknown repair pathway in vivo after genome editing. Really appreciated your suggestions. Nice weekend. Greetings, Guangzhi
To separate the sequences in the mix amplicon, you have to run new PCRs with one primer containing a base mismatch at 3' end
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