Professional Documents
Culture Documents
Experiment No1 - Pipetting Manual
Experiment No1 - Pipetting Manual
Experiment No. 1
Pipetting: Determine the protein concentration in a corn solution
I. Objectives:
To prepare different solution concentrations using a micropipettor and serial dilution
To determine the protein (amino acid) concentration in a genetically modifed corn solution using the Bradford
assay
II. Materials:
Phosphate buffer
Bradford reagent
Bovine Serum Albumin (BSA)
Corn (Zea mays)
Micropipettors
Spectrophotometer (Microplate Reader)
III. Procedure:
1. Obtain a sample of corn. (Zea mays)
2. Grind 100.0 g of corn sample with 10.00 mL of phosphate buffer using a mortar and pestle.
3. Filter the mixture and obtain the filtrate. Discard the residue.
4. Dilute the filtrate using a serial dilution. (Fill-up the table based on what you did in the simulation.)
Concentration Volume of solution Volume of buffer
1:5 uL of sample 800 µL
1:50 900 µL
1:500
1:5000
5. Prepare five standard solutions of 0.05, 0.10, 0.20, 0.30, 0.40, and 0.50 mg/mL of BSA in phosphate buffer.
6. Obtain 10 µL of each solution and transfer it into a well of a microplate following this arrangement:
A 1 (Sample - Trial 1) 2 (Sample -Trial 2) 3 (Standard – Trial 1) 4 (Standard – Trial 2)
B 1:5 1:5 0.50 mg BSA / mL 0.50 mg BSA / mL
C 1:50 1:50 0.40 0.40
D 1:500 1:500 0.30 0.30
E 1:5000 1:5000 0.20 0.20
F 0.10 0.10
G 0.05 0.05
7. Set the spectrophotometer (microplate reader) to 595 nmn.
8. Place the microplate inside the instrument.
9. Start the measurement and record the absorbance of each solution.
UST College of Science
Analytical Chemistry 1 Lab (CHM414L)
IV. Results:
V. Treatment of results
1. Get the average of each sample and standard solution.
2. Graph and perform a linear regression using the standard solution concentration (x) and average absorbance of
standards (y) to prepare a calibration curve. Determine the equation of the line.
3. Calculate the concentration of protein (in terms of BSA) of the sample solution within the calibration curve using the
equation of the line.
4. Calculate the amount of protein found in the corn sample in terms of mg/g of corn.