215-Lab8-PCR_Digests

215-Lab8-PCR_Digests - Lab 8 Restriction Digests & PCR...

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Lab 8 –Restriction Digests & PCR 8-1 Lab 8 Restriction Digests and PCR of Plasmid Samples The PCR-mediated mutagenesis of the Hst1::Sir2 chimera DNA fragment increases the frequency of mutations in the DNA. Most of the mutations will be base pair substitutions or single base pair insertions or deletions. However, it is also possible that larger deletions could be generated in the mutagenesis. These large deletions would also have a mutant phenotype in the silencing assays and would therefore pass through the screens that you have already performed. Although these mutations are defective in function, they will likely not provide much information on the relationship between the structure and function of the protein. We would therefore like to avoid the further characterization of these mutants if possible. To determine if any of your mutants contain large deletions you will perform restriction digests and PCR to examine the size of the Hst1:Sir2 chimera gene. In this lab you will set up the reactions. Next week you will run an agarose gel on these samples. Expt 8-1. Restriction Mapping of mutant plasmid DNA
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215-Lab8-PCR_Digests - Lab 8 Restriction Digests & PCR...

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