As a rule, avoid mixing different loci on the same gel : some markers need to be run longer than the others (see the spacing in the 100 bp ladder bands in Le Flèche 2006 Figure 2). Some are very easily typed, so that short runs are sufficient. Some markers require a 20 bp size marker, others a 100 bp size marker.
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May I run different loci on the same agarose gel ?
In the same section :
- I can get the PCR assay to work, but am having problems analyzing the bands to get accurate sizes from my gels. I wonder how to actually calculate an exact band size and the repeat number. I am using the size markers to make a standard curve based on distance migrated on the gel. This old fashioned method is working, but once I get a band size, I cannot figure out how to get a repeat copy number.
- Do I need to do a PCR on the control strain each time a PCR for an "unknown" strain is setup? Or could I simply use leftover product from a previous PCR on the control strain to run on a gel with the products from the strains I am trying to type ?
- I do not have the 16M reference strain. Is it possible to use an other control strain ?
- Is it better to use agarose or capillary electrophoresis for MLVA typing?
