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DNA Science Week 1

DNA Science Week 1 - – Extract certain DNA fragments(the...

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DNA Science Day 1 Amplifying and Cutting APh162 Winter 2005 Caltech
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So, what’s a plasmid?
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What are the tools? PCR = Xerox Machine Amplify DNA Restriction Enzymes = Scissors Target very specific DNA sequences Ligase = Glue Transformation and DNA extraction
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Making a modular plasmid Lutz and Bujard (1997) Copy number from 3 to 70 per cell Four possible antibiotic resistances Four promoters with three different inducers /HindIII
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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 – P LtetO-1 , repressed by the Tet repressor and induced by tetracyclin (ATC) Measure and compare the induction!
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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
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Unformatted text preview: – Extract certain DNA fragments (the vector) Digestion Protocol • Lambda/HindIII: – 3 ul Lambda/HindIII (1.5 ug) 5 ul NEBuffer EcoRI (10x) 40 ul ddH 2 O 2 ul EcoRI 50 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 Polymerase Chain Reaction 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 activation 2. 95C for 15 s – melting 3. 65C for 30 s – anheling 4. 68C for 3 min (min/kb) – elongation 5. Go back to 2 for a total of 35 cycles 6. Store at 4C • qPCR using SYBR green 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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