DNA Science Day 1 Amplifying and cutting科学日1放大与切割.pptVIP

DNA ScienceDay 1Amplifying and Cutting Physical Biology Bootcamp October 2006 Caltech Being Quantitative AboutGene Expression We can now answer a variety of questions quantitatively: The lac Operon, Where it all Began Modularity: Once the toolbox is developed you just need to shuffle the motifs to obtain novel behavior. The Players of the lac Operon So, what’s a plasmid? Quantifying gene expression Many ways of measuring gene expression LacZ activity GFP fluorescense mRNA level 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 Copy number from 3 to 70 per cell Four possible antibiotic resistances Four promoters with three different inducers The big picture Extract the lacZ gene from plasmid pZE21-lacZ (I extracted it originally from wild type type E. coli: MG1655, GenBank U00096). Put it into a pZS25 vector pSC101 origin of replication, ~10 copies. Kanamycin resistance PlacUV5, repressed by the Lac repressor and induced by IPTG. Measure and compare the induction! Doing a Restriction Digest Lambda DNA (NC_001416) predigested by HindIII. Show in Vector NTI. Go to the NEB site. We’ll digest it with EcoRI. Obtain our vector by digesting pZS25 plasmid with KpnI and HindIII. Show in Vector NTI Different looping lengths and sequences. 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 ~5 ug of your vector Just ~300 ng of DNA for the controls 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 (Accuprime II.pdf) Draw cycle! 1. 94C for 2 min – DNAP activation2. 94C for 15 s – melting3. 60C for 30 s –