Hi,
I use the primers (S926F and L189R) for ISR amplification by using PCR after the isolation of bacterial DNA. primers targeting the 3' end of the 16s and the 5' end of the 23s to amplify the 16s-23s rRNA Intergenic Spacer region.
I follow the instructions of the DNA polymerase for the cycle conditions but I couldn't get the product.
As I read I can separated the PCR product by using 1% agarose gel electrophoresis stained with Ethidium bromide.
There are too little details (i.e. what organism, DNA polymerase and primer structure do you use) to think about. Probably, the optimisation of annealing temperature will help. Optimal Ta for specific primers may differ from those in DNA polymerase manual. For example, if you used primers S926F\L189R from (Yu & Mohn, 2001) paper, your Ta should be 47C, but I am not sure that this case is relevant to your experiment.
Also, sometimes it is good idea to check the structure of primers that were ordered, as well as the quality of template DNA.
Kind regards,
Maxim
Thank you Maxim A Khasnatinov,
The annealing temp which I used was 58C, and according to DNA polymerase I used 10 uM for the concentration of the primers. These PCR cycling worked very well when I was working on 16s with 27F and 1492R but when I was working with these primers I found no products.
Thanks again,
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