dna science day 1 amplifying and cutting aph162 winter 2005 caltech
Post on 21-Dec-2015
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TRANSCRIPT
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
• 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