Cline Reaction Assay Protocol

1X Cline’s reagent: highest concentration (2g Diamine + 3g FeCl₃ in 50 mL 50% cool HCl)

For standard curve: weight out 240 mg of Na₂S₉H₂O, add to 100 mL 2.6% Zinc acetate. This bottle is 10 mM. The bottle should be prepared under anaerobic condition

Use a syringe to dilute 1 mL of 10 mM stock into 9 mL of 2.6% zinc acetate to make a 1mM stock, use for the following:

Final concentration

1 mM

0.75 mM

0.5 mM

0.25 mM

0.1 mM

0.075 mM

0.05 mM

0.025 mM

0.01 mM

0 Mm

mL of 1mM stock to add

1 mL

0.75 mL

0.5 mL

0.25 mL

0.1 mL

0.075 mL

0.05 mL

0.025 mL

0.01 mL

0 mL

mL of water

0 mL

0.25 mL

0.5 mL

0.75mL

0.9 mL

0.92 mL

0.95 mL

0.97 mL

0.99 mL

1 mL

Prepare a dilution of Cline’s reagent- “0.5X” by diluting 10 mL of the 1X reagent with 10 mL of 50% HCl (easiest way to do this would be to add 2.5 mL of HCl to 2.5 mL water, then add the reagent)

Place 30 μL of 1X Cline’s reagent into wells of a 96-well plate. Place 30 μL of 0.5X Cline’s reagent into wells of a 96-well plate.

Add 270 μL of sample to each well, mixing rapidly.

Allow 20 minutes for color to develop

Prepare a series of wells identical to the first, with 270 μL of DI water in them.

Transfer 30 μL of each developed sample to a well with water to dilute.

Read entire plate at 670 nm