Technical information
Centrifuge Standard Operating Procedure 24
and then fill with HS), cover centrifuge tube with parafilm and gently invert
1x.
13.Centrifuge @ 1,000 x g, 6 min, 4C in swinging bucket rotor (JS13.1, MAX
acceleration/deceleration).
14.Decant SN (can go down drain).
15.Re-suspend pellet in ~200-300 μl of HS buffer and transfer to 1.5 ml
microcentrifuge tube, while estimating the total volume (using pipette).
Part II: Chloroplast Concentration
1. Prepare reference sample:
a. 10 μl HS buffer
b. 990 μl 80% acetone
2. Prepare chloroplast sample:
a. 10 μl intact chloroplast suspension
b. 990 μl 80% acetone
3. Mix both samples by vortexing.
4. Centrifuge samples for 2 min @ max speed to pellet insoluble material.
5. Set the wavelength of the Cary 50 UV-Vis spectrophotometer to 652 nm, and
blank with reference sample.
6. Read absorbance of chloroplast sample at 652 nm.
7. Calculate chlorophyll concentration using the following equation:
chlorophyll concentration (mg/ml) = [A652/36] x DF;
where A652 is the absorbance of the sample measured at 652 nm and DF is
the dilution factor of the chloroplast sample (in this case the dilution factor
is 100).
8. Dilute chloroplasts to a chlorophyll concentration of 1 mg/ml.