In the interests of conserving time and resources, I would like to avoid performing replicates on every single sample, unless this is absolutely necessary. I have read in the literature that 5-10% of all samples can be sufficient for determining genotyping error in AFLP. Could a similar principle be applied to RAPD PCR?
SUBSET IS SUFFICIENT
Optimizations of the complete RAPD protocol, from the parameters upon which primer sequences are chosen to the conditions used for PCR, have been published (Williams et al., 1990; Nadeau et al., 1992). It is actually possible to obtain multiple PCR products with primers longer than 10-mers when relaxed reaction conditions are used to allow amplification from mismatched target sequences. In fact, one group has suggested that 12 to 14-mer primers are optimal (Nadeau et al., 1992). It is also possible to increase the predicted number of products by a simple factor of three by including two unrelated random primers of the same length in each PCR reaction. However, in any case where the number of visible PCR products goes above 12 to 20, it would become necessary to use polyacrylamide gels, rather than agarose gels, in order to clearly resolve each band; thus, the trade-off for the detection of more loci is a more time-consuming analysis. In the end, the protocol that requires the least amount of time for typing per locus is the one which should be chosen. Since different laboratories often excel at different techniques, the optimal conditions for RAPD analysis should be determined independently in each laboratory.
Sam, it depends of the journal you' re about to publish your data, but I've never read a paper in which the authors repeated the whole sample set only for making technical replicate (except in methodological journals). I would say that a subset of 5-10%, as you proposed, should be more than enough.
I agree with Branislave. Reproducible results with a subset of 10% will give you confidence about your result.
I agree with Branislave. Reproducible results with a subset of 10% will give you confidence about your result.
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