In my previous blog, I have described about amylose.
Today, I'll share about steps in amylose estimation, errors that might occur at each step and how to avoid.
1. Plotting Standard curve.
Standard curve is plotted using standard potato amylose starch which is pure amylose without amylopectin. 10 mg/ 10ml working solution is prepared by:
a. Weigh 10 mg potato amylose starch. Dissolve in 1 ml DMSO (Dimethyl Sulphoxide)
b. Vortex well
c. Incubate in waterbath at 85C for 10 mins.
d. Dilute it to 10 ml.
e. Pipette out 0, 20, 40, 60, 80, 100 microlitre of potato amylose starch into different tubes
f. Add 20 microlitre Iodine (I2:KI ) to each tubes.
g. Make final volume to 3 ml in each tube.
h. Mix well
i. Stand for 20 minutes
j. Take OD at 620 nm.
plot the values obtained graphically. A straight line is obtained.
Precautions: i. potato amylose starch should be accurately weighed.
ii. vortexing properly before incubating is necessary or else, the starch will gelatinize at the bottom.
iii. Pipetting should be clean and bubbles should be cleared while pipetting.
iv. Fresh standard curve should be plotted everyday, because pipetting varies day to day and person to person.
2. Samples estimation.
a. Weigh 20 mg sample.
b. Dissolve in 1 ml DMSO
c. Vortex properly.
d. Incubate in waterbath at 85C for 15 mins
e. transfer the 1 ml sample to fresh tube
f. Add 9 ml water.
g. From the 10 ml working solution, pipette out 100 microlitre aliquot into another test tube
h. Add 20 microlitre Iodine solution
i. Make up volume to 3 ml.
j. Incubate at RT in dark for 20 mins.
k Take O.D at 620 nm
Precautions:
i. Iodine is usually added after sample and water are added.
ii. The reaction is light sensitive hence should be kept in dark.
iii. The incubation period should not exceed the limit because the colour complex disappears.
iv. Iodine, sample and water should be properly mixed and should not remain on the walls of the test tube.
Usually 2-3 technical replicates and 2 biological replicates are done.
Today, estimated amylose from 50 samples.
Initially we used to estimate 25 samples in a single lot. The results were poor because by the time we would reach the last samples, their colour would disappear. Therefore we now carry out 12-13 samples in one lot and complete 50 samples in 4 lots.
That's all about procedure for amylose estimation.
Good night.
Today, I'll share about steps in amylose estimation, errors that might occur at each step and how to avoid.
1. Plotting Standard curve.
Standard curve is plotted using standard potato amylose starch which is pure amylose without amylopectin. 10 mg/ 10ml working solution is prepared by:
a. Weigh 10 mg potato amylose starch. Dissolve in 1 ml DMSO (Dimethyl Sulphoxide)
b. Vortex well
c. Incubate in waterbath at 85C for 10 mins.
d. Dilute it to 10 ml.
e. Pipette out 0, 20, 40, 60, 80, 100 microlitre of potato amylose starch into different tubes
f. Add 20 microlitre Iodine (I2:KI ) to each tubes.
g. Make final volume to 3 ml in each tube.
h. Mix well
i. Stand for 20 minutes
j. Take OD at 620 nm.
plot the values obtained graphically. A straight line is obtained.
Precautions: i. potato amylose starch should be accurately weighed.
ii. vortexing properly before incubating is necessary or else, the starch will gelatinize at the bottom.
iii. Pipetting should be clean and bubbles should be cleared while pipetting.
iv. Fresh standard curve should be plotted everyday, because pipetting varies day to day and person to person.
2. Samples estimation.
a. Weigh 20 mg sample.
b. Dissolve in 1 ml DMSO
c. Vortex properly.
d. Incubate in waterbath at 85C for 15 mins
e. transfer the 1 ml sample to fresh tube
f. Add 9 ml water.
g. From the 10 ml working solution, pipette out 100 microlitre aliquot into another test tube
h. Add 20 microlitre Iodine solution
i. Make up volume to 3 ml.
j. Incubate at RT in dark for 20 mins.
k Take O.D at 620 nm
Precautions:
i. Iodine is usually added after sample and water are added.
ii. The reaction is light sensitive hence should be kept in dark.
iii. The incubation period should not exceed the limit because the colour complex disappears.
iv. Iodine, sample and water should be properly mixed and should not remain on the walls of the test tube.
Usually 2-3 technical replicates and 2 biological replicates are done.
Today, estimated amylose from 50 samples.
Initially we used to estimate 25 samples in a single lot. The results were poor because by the time we would reach the last samples, their colour would disappear. Therefore we now carry out 12-13 samples in one lot and complete 50 samples in 4 lots.
That's all about procedure for amylose estimation.
Good night.
Potatoes amylose starch you are weighing 10mg and sample you are weighing 20mg, why this difference?
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