Hello to all,
I'm doing a master thesis on Campylobacter jejuni and I'm down to the genotyping, I'm using MLST and I obtained the first products (900 bp) however, they need to reduced to 500 bp using nested primers, whenever I run the pcr I get lots of non-specific bands, is there a way to genotype the isolates without the second pcr step, or are there a software that can reduce there sequence from 900 bp to 500 bp if I specify the primers ???
Thanks in advance
I don't think there is any software that can reduce the sequence from 900bp to 500bp. You might need to troubleshoot why there is presence of non-specific bands. Worse comes to worst, you may need to design a primer and re-start everything. Good luck.
Dear Ismail,
I think you should check your second primers specify in GenBank database. Also I suggest you should some modification in your second PCR amplification for avoid missed amplification products. After your MLST sequence analysis of different PCR products, you should upload your sequences in Bionumerics 7.6 software or other, you could find your MLST types of your agent. But I think you should check your second PCR primer specify and use some modification on your second PCR analysis (especially melting temperature of primers)
Another way to do that is you use nested primer as a sequencing primer to do sequencing the first PCR product.
But if you want to optimize your nested PCR, you can check primer and PCR condition in https://pubmlst.org/campylobacter/
Hi and thanks to all, I did everything according to the article and primers described by Kate E. Dingle, I have some solutions based on the answers you gave, now my question will be, can I genotype my isolates if I sequence the first products (the 900 bp bands) ???
Yes but you have to trim your data before submitting to database.
You can do primer blast for your nested primer and you'll see a sequence in between your nested primer and then you can use it as a reference for trimming. Hope this help.
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