Difference between revisions of "Part:BBa K1182000:Experience"

Revision as of 14:52, 26 September 2013

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===Applications of BBa_K1182000===

To test the function of our split reporter, we collect the data of enzyme activity over time. We exploit the enzyme’s ability to convert ortho-Nitrophenyl-β-galactoside (ONPG), a colorless substance, into ortho-nitrophenol, a yellow substance. We then use a spectrophotometer to quantisize the absorbance of the solution.

Experimental setup & protocol

1. The alpha and omega fragment of β-galactosidase was cloned into pQE-80L and pQE-81L, respectively

2. The alpha and omega fragment was then expressed in TOP10 E.coli

3. An equal molar of both the alpha and omega peptide, as well as the diluted full length β-galactosidase, then added into an eppendorf tube and incubated at room temperature on an orbital rocker for 1 hour.

4. At time zero, 20µL of ONPG (4mg/mL) was added into each tube

5. The eppendorf tubes then incubated at room temperature for different length of time (30 min, 90 min, 3 hours, and 19 hours).

6. The reaction was then terminated by adding 50µL 1M Na2CO3

7. The absorbance is the analysed using 420 nm light

Result

User Reviews

UNIQ6f7d233a6c7db4e9-partinfo-00000000-QINU UNIQ6f7d233a6c7db4e9-partinfo-00000001-QINU