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Problems Not all bacteria pick up plasmid-how do we distinguish? Remember ampR gene?

Problems Not all bacteria pick up plasmid-how do we distinguish? Remember ampR gene? Annealing of human DNA to plasmid is random-how do we distinguish which plasmids have human DNA? Remember location of restriction site Plasmids w/human DNA cannot breakdown lactose, therefore no blue color.

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Problems Not all bacteria pick up plasmid-how do we distinguish? Remember ampR gene?

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  1. Problems • Not all bacteria pick up plasmid-how do we distinguish? • Remember ampR gene? • Annealing of human DNA to plasmid is random-how do we distinguish which plasmids have human DNA? • Remember location of restriction site • Plasmids w/human DNA cannot breakdown lactose, therefore no blue color

  2. So what about our lab? • We are artificially transforming bacteria • Giving them a new gene-it will make them glow in the dark!!! • Gene comes from jelly fish! • Operon includes genes to make GFP AND IS controlled by arabinose, which is an inducer • How do we know which bacteria took up plasmid? • What is required in the agar to make them “glow”?

  3. SAFETY, SAFETY, SAFETY!!! • THINK! • Clamshell technique • Wipe surfaces thoroughly! • No food or drink anywhere!!!! • Don’t touch face, doorknobs, goggles, books, faucet handles, your ass,etc….

  4. How do we find the genewe want in a library?

  5. Sometimes there just isn’t enough Of the DNA we want to work with! The answer is PCR Polymerase Chain Reaction http://dnalc.bii.a-star.edu.sg/shockwave/pcranwhole.htm l

  6. http://www.pbs.org/wgbh/nova/genome/sequ_flash.html • DNA sequencing

  7. Southern Blotting http://dnalc.bii.a-star.edu.sg/shockwave/southan.htm

  8. Copy DNA- (cDNA) Since bacteria don’t normally Process their mRNA, they have no way of cutting out introns of eukaryotic genes

  9. DNA microarray analysis • Allows us to answer questions about gene activity • Different stages of development • Different tissues • Healthy vs diseased tissues

  10. RFLP’s-Restriction Fragment Length Polymorphismsnon-coding sequences have individual differences

  11. Alu insert

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