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In an attempt to analyze all 146 colonies without sequencing every one of them, PCR amplification was used. Since WRKY1 was obtained from the sequencing numerous times, it was assumed that a large proportion of the remaining unsequenced colonies may also contain WRKY1. DNA from the colonies was extracted from each yeast preparations and PCR primers designed to amplify the WRKY1 gene based on the shortest cDNA segment obtained from screening were used to identify WRKY1s amongst the un-sequenced colonies. All DNA extracted from colonies amplified at the expected WRKY1 300 base pair band except three from the Moroberekan and three from the C64 library. These six were sequenced. |
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