I want to amplify the entire gene. Does someone have a good protocol?
It depends on what it is you're trying to do. Regular pcr? If you have the sequence data for your region of interest, you can use free services like that of IDT or other softwares to design your own specific primers. Otherwise, you could order a couple of primers from published articles and test them for the best for your DNA sample. You don't necessarily have to amplify the entire gene, usually within the gene is good enough. Just make sure to verify the pcr product size as well as the amplified sequence (BLAST for instance). The size, if using primers from other publications must match the expected described, or if you designed your, then your expected based on how far apart the primers anneal. As for the protocol, if trying some from published articles, you should find the protocol described within the article. Otherwise, a similar protocol to that in a manuscript with the annealing temperature adjusted to match that for your primers should work just fine. (For example: 94C for 2min, 94C for 10sec, your annealing temp for 10secs, 72C for 20secs repeat step 2 to 4 33 times, 72C for 10min, 4C for ever.)
Rossana, one alternative is to acquire commercial reagents. I have been using Life tech. kits for 16S for a while and it works quite well. Your sequencing results will really look good. I also agree with Ken : you dont need to sequence the whole gene. I routinely use the Microseq 500 kit, which does the sequencing for the first 500 bp. They do have the option for the full gene kit, but we did a comparative analysis and for our application ( to identify atypical and unusual clinical bacterial isolates ) their performance is the same.
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