Transcript DNA Science Day 1 Amplifying and cutting

DNA Science
Day 1
Amplifying and Cutting
Physical Biology Bootcamp
October 2006
Caltech
Being Quantitative About
Gene Expression
• We can now answer a variety of questions
quantitatively:
How much?
When?
Where?
Small et al. (1992, 1996)
Elowitz et al. (2000)
Setty et al. (2003)
• Quantitative data demands quantitative
models! In vivo modeling is needed.
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
Tom Kuhlman and Terry Hwa
• Does the message depend on the messenger?
– Are the different reporters linear with respect to each
other?
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
/HindIII
• Copy number from 3 to
70 per cell
• Four possible antibiotic
resistances
• Four promoters with
three different inducers
Lutz and Bujard (1997)
5 PlacUV5
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 ddH2O
2 ul EcoRI
50 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 activation
2. 94C for 15 s – melting
3. 60C for 30 s – anheling
4. 68C for 3.5 min (min/kb) – elongation
5. Go back to 2 for a total of 35 cycles
6. Store at 4C
Gel Electrophoresis
• Preparing the samples:
– <150 ng
– Loading dye
– DNA ladders (pg. 10)
• Run 1% TAE gel at 90 V for ~80 min
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