ProblemsProblemsNot all bacteria pick up Not all bacteria pick up plasmid-how do we plasmid-how do we distinguish?distinguish?

Remember ampR Remember ampR gene?gene?

Annealing of human Annealing of human DNA to plasmid is DNA to plasmid is random-how do we random-how do we distinguish which distinguish which plasmids have human plasmids have human DNA?DNA?

Remember location of Remember location of restriction siterestriction site

Plasmids w/human Plasmids w/human DNA cannot breakdown DNA cannot breakdown lactose, therefore no lactose, therefore no blue colorblue color

So what about our lab?So what about our lab?We are artificially We are artificially transforming bacteriatransforming bacteria

Giving them a new gene-it will Giving them a new gene-it will make them glow in the dark!!!make them glow in the dark!!!

Gene comes from jelly fish!Gene comes from jelly fish!

Operon includes genes to Operon includes genes to make GFP AND IS make GFP AND IS controlled by arabinose, controlled by arabinose, which is an inducerwhich is an inducer

How do we know which How do we know which bacteria took up plasmid?bacteria took up plasmid?

What is required in the agar What is required in the agar to make them “glow”?to make them “glow”?

SAFETY, SAFETY, SAFETY!!!SAFETY, SAFETY, SAFETY!!!

THINK!THINK!

Clamshell techniqueClamshell technique

Wipe surfaces thoroughly!Wipe surfaces thoroughly!

No food or drink anywhere!!!!No food or drink anywhere!!!!

Don’t touch face, doorknobs, goggles, Don’t touch face, doorknobs, goggles, books, faucet handles, your ass,etc….books, faucet handles, your ass,etc….

How do we find theHow do we find the gene genewe want in a library?we want in a library?

http://dnalc.bii.a-star.edu.sg/shockwave/pcranwhole.html

Sometimes there just isn’t enoughOf the DNA we want to work with!

The answer is PCRPolymerase Chain Reaction

http://www.pbs.org/wgbh/nova/genome/sequ_flash.htmlhttp://www.pbs.org/wgbh/nova/genome/sequ_flash.html

DNA sequencingDNA sequencing

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

Southern Blotting

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

DNA microarray analysisDNA microarray analysis

Allows us to answer Allows us to answer questions about gene questions about gene activityactivity Different stages of Different stages of

developmentdevelopment Different tissuesDifferent tissues Healthy vs diseased Healthy vs diseased

tissuestissues

RFLP’s-Restriction Fragment Length PolymorphismsRFLP’s-Restriction Fragment Length Polymorphismsnon-coding sequences have individual differencesnon-coding sequences have individual differences

Alu insertAlu insert