DNA ScienceDay 1

Amplifying and Cutting

APh162Winter 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

Lutz and Bujard (1997)

• Copy number from 3 to 70 per cell

• Four possible antibiotic resistances

• Four promoters with three different inducers

/HindIII

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

• 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

• 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 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 – 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• 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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