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 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, copies. –Kanamycin resistance –P LtetO-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 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

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 -+