Genotyping Project Setup
- Make an appointment with Oanh for orientation in genotyping with ABI instruments/software
- Topics we'll go over:
- Sample submission process
- Genotyping with capillary electrophoresis
- GeneScan and GeneMapper introduction
- Genotyping protocols and ABI compatible kits
- Multiplexing reactions to save on the number of capillaries used
- Sequence a set of "control" samples for the sizing project.
- Sequenced controls will show the actual number of repeats. This number should then be used to analyze GeneScan sizing data. The two numbers (one produced by sequencing, the other with GeneScan) will almost always differ. The difference in size calling is caused by unavoidable mobility differences between the 2 platforms, but is reproducible.
- After performing the fluorescence emissivity tests(see #3) for all your template and primer combinations, youshould include a sequenced control in every set of 16 capillaries you will be submitting for the duration of the sizing project.
- Sequenced controls provide an additional internal standard to identify any unusual mobility irregularities that may occur from run to run. (One "run" consists of 16 capillaries.)
- Submit a set of dye-labeled PCR products as a dilution series in fluorescence emissivity testing for every template and primer combination you are interrogating.
- Complete the on-line Service Request for the dilution series.
- Generally, performing this test once for a particular template and primer combination should be sufficient, assuming your PCR conditions do not change.
- It is absolutely necessary to prevent overloading of the capillaries.
- Overloaded capillaries produce large, broad peaks that the instrument is incapable of sizing reliably.
- PCR products should produce fluorescence values in the range of 1000 to 3000 rfu's (relative fluorescence units-an ABI term)
- Since you will be identifying a general dilution factor for every template and primer combination, there may be instances where signal will be insufficient or too strong for particular samples. The "fix" for these is altering the dilution factor appropriately and submitting a new service request for the samples in question.
- Series dilution suggestions
- Pick 4 random samples representing a particular template and primer combination for dilution testing
- Perform a dilution series for each template/primer combo such as 1:5, 1:10, 1:20, and 1:50
- Decide which dilution factor produces rfu's between 1000 and 3000 for the majority of your samples.
