DNA Science Day 1 Amplifying and Cutting
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DNA ScienceDay 1
Amplifying and Cutting
APh162Winter 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– PLtetO-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– Extract certain DNA fragments (the vector)
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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
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Polymerase Chain Reaction
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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 – 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
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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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