Lec14_w11

Lec14_w11 - Vibrio fischeri DNA isolation Create genomic...

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Vibrio fischeri DNA isolation Create genomic library in pGEM and DH5 α Screen for expression of bioluminescence genes Pick glowing colony, expand in liquid culture, and isolate plasmids Digest plasmid from glowing colony, isolate fragment containing just luxA and luxB Screen for non-glowing white colonies Pick 2 white colonies, expand in liquid culture, and isolate plasmids Sequence insert in plasmids Subclone luxA and luxB into pUC18 and transform DH5 α Generate PCR product containing luxA and luxB Identify gene using Bioinformatics tools Add aldehyde and screen for bioluminescence
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1. Review Sanger sequencing and ORF finder http://www.dnalc.org/resources/animations/ Orf finder -http://www.ncbi.nlm.nih.gov/projects/gorf/
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Longest open reading frame is 507 nucleotides; the reading frame is -1 That sequence translated into amino acid sequence
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Your DNA sequence after translation by ORF finder The sequence of the best match in Genbank – in this case, ABC transporter membrane protein from Vibrio fischeri
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Sac 1 leaves a sticky end Hinc ll and Eco RV leave blunt ends Take PCR product – digest with Sac
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This note was uploaded on 04/10/2011 for the course BIMM 101 taught by Professor Butler during the Spring '08 term at UCSD.

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Lec14_w11 - Vibrio fischeri DNA isolation Create genomic...

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