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DNA Science Day 1 Amplifying and Cutting

DNA Science Day 1 Amplifying and Cutting. Physical Biology Bootcamp October 2006 Caltech. Being Quantitative About Gene Expression. Small et al. (1992, 1996). Elowitz et al. (2000). Setty et al. (2003). We can now answer a variety of questions quantitatively:. How much?. When?.

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DNA Science Day 1 Amplifying and Cutting

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  1. DNA ScienceDay 1Amplifying and Cutting Physical Biology Bootcamp October 2006 Caltech

  2. Being Quantitative AboutGene Expression Small et al. (1992, 1996) Elowitz et al. (2000) Setty et al. (2003) • We can now answer a variety of questions quantitatively: How much? When? Where? • Quantitative data demands quantitative models! In vivo modeling is needed.

  3. The lac Operon, Where it all Began • Modularity: Once the toolbox is developed you just need to shuffle the motifs to obtain novel behavior.

  4. The Players of the lac Operon

  5. So, what’s a plasmid?

  6. Quantifying gene expression Tom Kuhlman and Terry Hwa • Many ways of measuring gene expression • LacZ activity • GFP fluorescense • mRNA level • Does the message depend on the messenger? • Are the different reporters linear with respect to each other?

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

  8. 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 PlacUV5

  9. The big picture • Extract the lacZ gene from plasmid pZE21-lacZ (I extracted it originally from wild type type E. coli: MG1655, GenBank U00096). • Put it into a pZS25 vector • pSC101 origin of replication, ~10 copies. • Kanamycin resistance • PlacUV5, repressed by the Lac repressor and induced by IPTG. • Measure and compare the induction!

  10. Doing a Restriction Digest • Lambda DNA (NC_001416) predigested by HindIII. • Show in Vector NTI. • Go to the NEB site. • We’ll digest it with EcoRI. • Obtain our vector by digesting pZS25 plasmid with KpnI and HindIII. • Show in Vector NTI • Different looping lengths and sequences. • Run the results on an agarose gel. • Analyze our results. • Extract certain DNA fragments (the vector).

  11. 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 ~5 ug of your vector • Just ~300 ng of DNA for the controls • Let sit for 2-3 hours at 37ºC

  12. Polymerase Chain Reaction

  13. Polymerase Chain Reaction • Designing a primer • Adding a couple of sites • (APh162 Primer.doc) • The components and protocol • (Accuprime II.pdf) • Draw cycle! • 1. 94C for 2 min – DNAP activation2. 94C for 15 s – melting3. 60C for 30 s – anheling4. 68C for 3.5 min (min/kb) – elongation5. Go back to 2 for a total of 35 cycles6. Store at 4C

  14. Gel Electrophoresis • Preparing the samples: • <150 ng • Loading dye • DNA ladders (pg. 10) • Run 1% TAE gel at 90 V for ~80 min - +

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