For the identification of Biosurfactant producing bacteria, I am trying to sequencing the 16s rDNA, that i had done three time. Unfortunately, i did not get any results. After amplification, i have found goodconcentration and purity.
Did you check your DNA ??
Do a PCR with any lab grown bacterial DNA to check your primers
Next, BLAST your primers seq. with whole genome of your desired bacterial strain.
Three common reasons for pcr not working are poor template quality, poor primers and wrong pcr parameters. Abhijit is correct that you need a good dna sample which preferably has amplified before with other primers…borrow from colleagues if necessary, then make a few dilutions of dna and amplify with 2.5mM magnesium and 35 cycles on a gradient of annealing temperatures and twice as much primer as you are using now. You should get amplification but probably quite dirty but it is easier to clean up a pcr when it is working than it is to find out what the problem is when you are getting no product. If this approach fails the it is probably time to order new primers.
You say you get your DNA amplified but you don't get results from sequencing?
Do you re-measure your DNA after the clean-up step before/after the sequencing reaction? Maybe the DNA is being lost during one of the clean-up steps.
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