PrimeSTAR GXL DNA Polymerase Amplifies Genomic DNA from Crude C. elegans Extracts

For this project, my goal was to use a PCR-based strategy to genotype C. elegans strains in order to confirm the presence or absence of specific mutations. Because I was genotyping anywhere from 4-16 strains for a given mutation, we wanted a protocol that would allow us to use crude extracts made from whole C. elegans samples. The DNA polymerase we used would have to be a robust enzyme that could amplify its target from extracts that contained cellular proteins and other molecules in addition to the target genomic DNA. We first tested several polymerases and found that they failed to amplify any products. To troubleshoot, we decided to test Takara’s PrimeSTAR GXL DNA polymerase because this polymerase is advertised as being able to amplify PCR products from GC-rich templates, in the presence of excess nucleic acid, and when the target is a long (multi-kilobase) sequence. PrimeSTAR GXL DNA polymerase gave us robust, specific results that allowed us to deduce the genotypes of our strains. We amplified five different genomic targets within four different genes. For one gene, we used the PCR product to perform DNA sequencing, which worked on our first attempt. Sometimes it was necessary to test more than one annealing temperature to optimize a PCR reaction, but we did not need to perform additional optimization reactions.