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Amplifying and Cutting DNA: A Comprehensive Guide to Plasmid Design and Analysis

This resource provides an in-depth exploration of fundamental molecular biology techniques used in DNA amplification and manipulation. It covers essential tools such as PCR, restriction enzymes, and ligases in the construction of modular plasmids, allowing for antibiotic resistance selection and controlled gene expression. Step-by-step protocols include digesting Lambda DNA, designing primers, and analyzing digestion results on agarose gels. Ideal for students and researchers, it provides a practical overview of cloning and transformation processes, underscoring the importance of precision and technique in genetic engineering.

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Amplifying and Cutting DNA: A Comprehensive Guide to Plasmid Design and Analysis

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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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