What is the best procedure for sequencing bacteria in phylogenetic analysis. Using isolated plasmid DNA from the bacterial cells and adding omto the primer (27F and 1492R) or just taking the sample directly from the cells in the LB media and adding onto the primers (27F and 1492R)??
Take the bacterial cell samples from the LB media and add directly onto the primers (27F and 1492R) for the PCR amplification and follow as suggested by Dr. Khem Raj.
Regards
get the forward and reverse sequence of the product followed by merge by using online software. now use this merge sequence for the phylogenetic analysis. this can be draw either Bioedit software or mega software. You can also use directly CLC workbench software if available
Dr. Khem
@Khem Raj Meena. I understand your explanation but i wanted to know the best approach from the initial PCR amplification stage. Should I use the isolated plasmid DNA from the bacterial cells and add onto the primer (27F and 1492R) or just take the bacterial cell samples from the LB media and add directly onto the primers (27F and 1492R) for the PCR amplification??
Take the bacterial cell samples from the LB media and add directly onto the primers (27F and 1492R) for the PCR amplification and follow as suggested by Dr. Khem Raj.
Regards
Alternatively you can draw samples from 24 hour LB broth culture, Centrifuge and discard the medium. Add approximately 100 microlitre ddH2O and vortex then lyse the cells through boiling, cool for 1 min, centrifuge once more then use the supernatant as template for amplification of the 16s rRNA gene region using the stated universal primers.
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