Hi
I extracted plasmid from B cereus (isolated from rice in which emetic strains r more prevalent)
I am trying to detect emetic toxin (CES) gene which is located on plasmid.
When I run gel to see the ces gene, I only see bands for only three samples out of 38 (one of which is the positive control, emetic B cereus DSMZ 4312).
I then run gel to see plasmid DNA but none were visible including that of positive control n other two samples which gave bands of ces gene amplicon.
It' s strange that plasmid DNA is not visible for all samples but when amplified, certain samples give bands indicating amplification of genes on the plasmid ( I put 1ul of dna template in 25ul PCRmixture)
Assuming the DNA is too dilute (when not visible on gel), I increased the volume added to 3ul of DNA template, it still gives similar results..
Im really confused
Your assays are from cultivation or sample?
If it is from samples, first analyze the primers (you can use PrimerBlast), verify if the size of the predicted primers corresponds with those of your gel.
If it is from culture, you are probably having a problem with the extraction (you can increase the cell concentration), usually using liquid medium from which you concentrate. there must be more than 100 million per milliliter for a good recovery if you are doing by in-house methods.
Greetings and success.
Luis A Salcedo, I extracted plasmid from B cereus culture (grown in LB broth).
Md Abdur Rahim, I used the commercial plasmid extraction kit.
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