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DNA Science Day 1 Amplifying and Cutting

DNA Science Day 1 Amplifying and Cutting. APh162 Winter 2005 Caltech. So, what’s a plasmid?. What are the tools?. PCR = Xerox Machine Amplify DNA Restriction Enzymes = Scissors Target very specific DNA sequences Ligase = Glue Transformation and DNA extraction. Making a modular plasmid.

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DNA Science Day 1 Amplifying and Cutting

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  1. DNA ScienceDay 1Amplifying and Cutting APh162 Winter 2005 Caltech

  2. So, what’s a plasmid?

  3. What are the tools? • PCR = Xerox Machine • Amplify DNA • Restriction Enzymes = Scissors • Target very specific DNA sequences • Ligase = Glue • Transformation and DNA extraction

  4. Making a modular plasmid • Copy number from 3 to 70 per cell • Four possible antibiotic resistances • Four promoters with three different inducers /HindIII Lutz and Bujard (1997)

  5. The big picture • Extract the lacZ gene (our insert) from wild type E. coli (MG1655, GenBank U00096). • Put it into a pZE21 vector • colE1 origin of replication, 60-70 copies. • Kanamycin resistance • PLtetO-1, repressed by the Tet repressor and induced by tetracyclin (ATC) • Measure and compare the induction!

  6. Doing a Restriction Digest • Lambda DNA (NC_001416) predigested by HindIII • Go to the NEB site • We’ll digest it with EcoRI • Obtain our vector by digesting pZE21-GFP with KpnI and HindIII • Run the results on an agarose gel. • Analyze our results • Extract certain DNA fragments (the vector)

  7. Digestion Protocol • Lambda/HindIII: • 3 ul Lambda/HindIII (1.5 ug)5 ul NEBuffer EcoRI (10x)40 ul ddH2O2 ul EcoRI50 ul Total • Double Digest: • Start the cloning with ~3 ug of your vector • Just ~300 ng of DNA for the controls • (pg. 17) • Let sit for 2-3 hours at 37ºC

  8. Polymerase Chain Reaction

  9. Polymerase Chain Reaction • Designing a primer • Adding a couple of sites • (APh162 Primer.doc) • The components and protocol • (InvitrogenAccuprimePFXSupermix.pdf) • Draw cycle! • 1. 95C for 5 min – DNAP activation2. 95C for 15 s – melting3. 65C for 30 s – anheling4. 68C for 3 min (min/kb) – elongation5. Go back to 2 for a total of 35 cycles6. Store at 4C • qPCR using SYBR green

  10. Gel Electrophoresis • Preparing the samples: • <150 ng • Loading dye • DNA ladders (pg. 10) • Run 1% TAE gel at 100 V for ~80 min - +

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