Standard Protocols

Luciferase Assay

Make the following stock solutions.

1M HEPES pH 8 (RT)

1M DTT (4ºC)

100mM MgCl2 (RT)

1M Glycylglycine pH8 (4ºC)

500mM EDTA (RT)

Reporter Lysis solution

10ml 1M HEPES ph8

0.5ml 1M DTT

2ml 100mM MgCl2

2ml Triton X100

85ml distilled water

Luciferin 10mM stock solution

50mg Beetle Luciferin (promega E160B)

0.47ml 1M Glycylglycine ph8

15.3ml distilled water.

(Aliqouts are kept in bob’s freezer draw)

Luciferase Assay Reagent (LAR)

2ml 1M Glycylglycine

1ml 100mM MgCl2

20 ul 500mM EDTA

50.8mg DTT

27.8mg ATP

21.3mg coenzyme A (Sigma C3019)

Add 250m l aliquot of Luciferin stock to each 5mls of LAR

For long term storage of luciferase assay reagent it is recommended that aliqouts of 5mls are stored at –70ºC

1 Transfect cells in the usual way.

2. Remove media and wash with PBS.

Add enough luciferase lysis solution to the cells to cover them i.e. 100m l/well of a 96 well plate.

3. Freeze/Thaw at –70ºC. Cells can be stored for upto 2 weeks at –70ºC before assaying.
4. Transfer a 50m l sample of lysate to a rohen tube.
5. Add 250m l aliquot of Luciferin 10mM stock to 5mls of luciferase Assay Reagent .
6. Add 100m l of luciferin/luciferase solution to each sample and measure in luminometer or plate reader downstairs.

Research @ Birmingham

Gene Therapy