Lab 2 Flashcards
Thermostable DNA polymerase
- Also called Taq from thermophilic bacteria , Thermus aquaticus
What is Taq polymerase used for
PCR
PCR
- polymerase chain reaction : A technique used to make millions of copies of a particular section of DNA
What is the rate of Taq polymerase
- Makes DNA 3000 bases/ min with at least 1 mistake per 10,000 bases
What is the purpose of this lab ?
To purify recombinant Taq DNA polymerase from E Coli
What are the advantages of using E Coli vs T aquaticus ?
- Easier to grow
- grows better in liquid culture
- faster duplication time
- can be engineered to overexpress Taq protein
- easier to purify since it is not thermostable
The gene encoding Taq DNA polymerase is inserted into a plasmid called
pAKTaq
What gene sequences does the pAKTaq plasmid contain ?
- Ampicillin resistance gene ( Amp R)
- lacl ( lac repressor) gene
- ORC
Taq gene controlled by Lacl protein
What enzyme does the ampicillin resistance gene encode
- Enzyme β-lactamase, which allows for bacteria to grow in the presence of ampicillin by cleaving the lactam ring which usually makes them unable to grow
When the Lacl repressor is bound to the lac operator
- RNA polymerase cannot bind to promotor region
- no transcription and gene is turned off
How can the lac l repressor gene be turned on?
- Adding lactose or IPTG which bind to Lacl repressor
- Lacl repressor leaes operator sequence allowing RNA polymerase to ind
- gene transcription
- Gene is turned on
IPTG
molecular biology reagent that mimics allolocatose , a lactose metabolite that triggers transcription
What are the stages of bacterial growth ?
- lag phase
- exponential phase
- stationary phase
- death phase
Why should E Coli cells be allowed to grow without IPTG till they reach the exponential growth phase ?
- overexpression of the Taq protein is toxic for E Coli
- IPTG will induce the expression of the protein
At what wavelength absorbance is the IPTG being added
650 nm
