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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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Presentation Transcript
what are the tools
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
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)

the big picture
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!
doing a restriction digest
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)
digestion protocol
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
polymerase chain reaction1
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
gel electrophoresis
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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