I cultured Bifidobacterium animalis, identified on MALDI-TOF and later introduced it into broth. I cultured it for 48 hours and then extracted DNA from it using the Masterpure DNA kit with slight modification (beat beating step + cocktail of enzyme lysozyme, mutanolysin and lysostaphin). However when i ran the PCR and checked the PCR products on 1% agarose gel, i did not get any band. i have tried different concentrations. It is not a problem with my primers has this has been shown to work for my positive control. I am not sure what i am doing wrongly. I have attached a gel image of the PCR products
Hi
i think the problem comes from your DNA preparation and one way to find out is to mix you positive controle with one eluted DNA and perfome PCR reaction. If you have some PCR inhibitors your PCR will give you a negative resulte and from there you have to clean you DNA
I hope this will help
Ok. Thank you Abderrahmane. I will try that and let you know.
Dear
Firstly, you must confirmed that a growth was occurred through the turbidity appearance. Secondly, The presence of DNA ladder indicate no problem in PCR amplification and good agarose gel electrophoresis. I recommend to re-extract the DNA from the sample and if you available, Samaga automated bacterial DNA extraction Unit is highly recommended.
Hi all.
Thanks for your help. I discovered i was using too much of my DNA template for running the PCR. Also i had to clean up my DNA using PCR inhibitor removal kit from zymoresearch. It worked well after this and i could see my visible bands on gel. @Abderrahmane Bengrine
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