I've designed a novel Multiplex-T RFLP protocol for the simultaneous detection of a various strains of pathogens from a genus of bacteria. The multiplex design and gene selection mean that each strain of bacteria produces a unique pattern of amplicons of different length. I'm then using T-RFLP to visualise these fragments on an electropherogram, allowing detection of different strains simultaneously. As only the 3' of each amplicon is labelled with a fluorescent dye, is restriction digestion prior to T RFLP necessary?
Hi Artur, I agree by definition, you're right it won't be RFLP. I guess my question is more of a technical question as to whether the sequencing machine (a capillary-based Applied Biosystems AB3730) can detect and measure entire labelled amplicons without prior amplicon digestion?
it depends a little on what length capillary you use but the size standards are good for fragments up to 1000 bases but it does depend a little on the size differences between the sorted fragments
Hi Paul, thanks for your advice. All amplicons should be under 1000 bp, and have sufficiently different expected lengths to allow discrimination between fragments. I'll submit a plate and see! Thanks
Check first that the restriction is not essential in the process. For instance if 2 organisms both amplify 200 base amplimer but digestion cuts one organism to distinguish it from others then just the full length amplimer may be of limited use but if amplimer size defines your organism then you are fine.
best wishes
Paul
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