Materials and Methods LR2

Materials and Methods LR2 - Materials and Methods Isolation...

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Materials and Methods Isolation of LuxAB gene by PCR. Using the Polymerase Chain Reaction amplify the luxAB genes from V. fischeri DNA. Run products through gel electrophorese to confirm successful amplification. Clean up PCR products by MinElute PCR Purification Kit. Purifying plasmid DNA from previous colonies and preparation for final digestions. Obtain culture samples from one blue colony, two non-glowing white colonies, and one glowing white colony and grow in LB tube. Using Qiagen miniprep kit, purify the samples of plasmid DNA obtained from the colonies. Perform digestions on purified plasmid DNA using the restriction enzyme SalI. Run digested plasmid DNA against un-digested plasmid DNA in gel electrophoreses to confirm successful 9kb SalI fragments cloned into plasmid pGEM of glowing white colonies. Restriction digests of lux operon to isolate LuxAB gene. Add restriction enzymes SacI/EcoRV to plasmid pGEM and PCR products for digestion.
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