1.Spinach leaves 30 grams.
2.Knife and scissors.
7.50 ml centrifuge tubes.
8.1.5 ml centrifuge tubes.
Reagents and buffers
1. 1x Chloroplast isolation buffer without BSA:- 0.33M sorbitol, 0.1M tris-Cl ph 7.8, 5mM MgCl2, 10mM NaCl, 2mM EDTA.
2. 1x Chloroplast isolation buffer with BSA (0.1%w/v):
3. 40% percoll: 4ml percoll and 6 ml 1x CIB buffer with BSA to make 10 ml of 40% percoll. (Use 10 ml of 40% percoll for 6ml of chloroplast suspension)
4. 80% acetone.
1. Wash 30 gms of spinach leaves thoroughly first with tap water and then with distill water.
2. Remove the midrib veins of the leaves and cut into small pieces.
3. Add 120 ml of 1xCIB buffer with BSA to the cut leaves in a blender. Blend with 2-3 strokes.
4. Filter the blended leaves through 6 layers of muslin cloth.
5. The filtrate is then evenly divided into four 50 ml centrifuge tubes.
6. Centrifuge the tubes for 3 minutes at 200xg. A white pellet will be obtained.
7. Transfer the supernatant into chilled 50 ml centrifuge tubes and centrifuge at 1000xg for 7 minutes. A green pellet will be obtained.
8. Discard the supernatant and break the green pellet gently by finger tapping.
9. Resuspend the pellet in 2ml of 1x CIB buffer with BSA and mix gently by pipetting up and down.
10.Pool the suspended pellet into one centrifuge tube.
11.Preparation of 40% percoll layer: Mix 4 ml percoll with 6 ml of 1x CIB buffer with BSA.
12.Gently overlay 6ml of the chloroplast suspension over this 40% percoll layer.
13.Centrifuge at 1700 xg for 6 minutes. The intact chloroplast will sediment to the bottom of the tube as a green pellet and the broken chloroplast will form the upper layer.
14.Carefully remove the upper layer of the chloroplast suspension leaving only the pellet containing the intact chloroplast.
15.Mix the pellet with 500 ul of 1x CIB buffer without BSA.
Estimation of chlorophyll concentration
16.Add 10 ul of chloroplast suspension to 990ul of 80% acetone solution and mix gently.
17.Centrifuge at 3000xg for 2 minutes.
18.Take 100ul of the supernatant and transfer into a cuvette and measure the absorbance at 650 nm. Use 100 ul of 80% acetone as blank.
19.Take duplicate OD 650 values.
20.Take the average of the two values and estimate the mg/ml chlorophyll concentration using the following formula:
A 650 x 100/36 = mg/ml chlorophyll.
Where A 650 is the absorbance at 650 nm, 100 is the dilution factor and 36 is the extinction coefficient of chlorophyll.
Difference Encountered in a Real Laboratory
In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:
1. Before starting the experiment sterile the laminar air flow chamber using spirit.
2. Always disinfect your work area when you are finished.