After 16S rRNA gene sequencing, I'm trying to design primers to amplify a specific gene before proceed random Mutagenesis. Whole genome sequencing is not possible in my current situation. The information that I have with me is the species identification from the 16s rRNA gene sequencing results. What are the options that are available for me to proceed from here?
Dear Patrick, firstly you can find your desired full length or partial gene sequences from GenBank related with your microorganisms. Than you can design primer set using web based Primer3 program. However, the best way usage of primers described previous papers.
Best regards.
Download reference seq from:
http://www.ensembl.org/index.html
and then design primers using:
http://bioinfo.ut.ee/primer3-0.4.0/primer3/
I agree with @Muhammad Umair if you have genomic sequence for your target then primers3 is the best option for selecting sequencing primers.
Try to get as many lipase gene sequences from Genbank (especially related to your organism or near relatives). Align the sequences, then find the conserved regions (strict or majority rule consensus sequence). Compare the sequences with the consensus and try to design primers for highly conserved regions. It could be necessary to include wobbles.
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