FACULTY OF RESOURCE SCIENCE AND TECHNOLOGY DEPARTMENT OF MOLECULAR BIOLOGY STB1083-BIOCHEMISTRY
Practical 1 Title of experiment: Dilution Experiment DATE OF EXPERIMENT GROUP
MEMBERS
MATRIC NUMBERS
: 03/24/15 & : Lai Kim Yen
47236
Lau Sin Yee
47255
Lingeshwarry a/p Jewarethnam
47429
Maizatul Zulaikha Binti Azli
47491
Mariani Kelai Sanggau
47507
LECTURER
: Dr.Mickey Vincent
REPORT DUE DATE
: 03/31/15
INTRODUCTION
The initial part of being in a laboratory is to know the safety requirements and skills. It is crucial because if one do not have the skills, it would be difficult to carry out the experiment safely. Since solutions are usually highly concentrated than desired or can be managed based on a given protocol, it is often necessary for the dilution process to be performed on the solutions in order to get the desired level. This requires knowledge of the principle and procedures regarding the dilutions and even the calculation involved. Dilution is the process of combining measured volumes of a concentrated solution of analayte with a buffer to make less concentrated solution so that the concentration of the solute becomes lower. It is a process repeated constantly in the laboratory. There are several ways to make dilutions and one of them is the serial dilutions.
OBJECTIVE •
To be familiarize oneself towards the equipments and procedures involved in the serial dilution process
•
To be able to learn, perform and operate serial dilution processes
•
To carry out calculations such as concentrations and dilution factor based on the serial dilution processes
MATERIAL 0.5% (W/V) starch solution Iodine Distilled Water Glass Pipets Test Tubes Test Tube Rack
METHODS 1. 9ml of distilled water was placed into 5 test tubes using a glass pipet. 2.1ml of 0.5% (w/v) starch solution blue in the test tubes was taken and added into the test tubes containing 9ml of distilled water. The solution was mixed well and left to settle. It was labeled A. 3.1ml of test tube A was transferred into the next test tube containing 9ml of distilled water. The solution was mixed well and labeled B. 4.1ml of test tube B was transferred into the next test tube containing 9ml of distilled water. The solution was mixed well and labeled C. 5.1ml of test tube C was transferred into the next test tube containing 9ml of distilled water. The solution was mixed well and labeled D. 6.1ml of test tube D was transferred into the next test tube containing 9ml of distilled water. The solution was mixed well and labeled E. 7.An aliquot of iodine solution was added to all test tubes and the observation was recorded and described. 8. The starch concentration and dilution factor was calculated and tabulated for test tubes A to E.
RESULT AND CALCULATION: Test tube A: (Weight / Volume) x 100 % = 0.5% (Weight / 1ml) x 100 %=0.5% Weight =0.005g = 5mg =500μg X= 500μg/ml =starch concentration for test tube A Test tube B: M1V1=M2V2 (500)(1)= M2 (10) M2= 50μg/ml Test tube C: M1V1=M2V2 (50)(1)= M2 (10) M2=5μg/ml Test tube D: M1V1=M2V2 (5)(1)= M2 (10) M2= 0.5μg/ml Test tube E: M1V1=M2V2 (0.5)(1)= M2 (10) M2=0.05μg/ml Test tube
Starch concentration
Dilution factor
A B C D E
(μg/ml) 500 50 5 0.5 0.05
1: 10 1:100 1:1000 1:10000 1:100000
DISCUSSION Before entering the biochemistry laboratory, there is some basic but vital safety that had to be followed. First and foremost, everyone should wear a lab coat, glove and fully-cover shoe as to prevent any indirectly spilt of chemical towards our body. From this experiment, we will able to graph analyse the experiment data through the spreadsheet. Besides plotting on the graph in the graph paper, we also can use Microsoft excels to plot the graph. First, enter and format data in the Excel spreadsheet, then directly create a scatter plot from the spreadsheet data and remember to label the x-axis and y-axis as well as the title of the graph as to let the other to understand the information of the graph. Next, a trendline feature of Excel can help in finding the best fit line for the data information. By using the Excel to plot a graph make it fast and easy to insert the data to spreadsheets as to provide a visual presentation. Another laboratory technique included the metric system which refers to an internationally agreed decimal system of measurement. For example, in this experiment, the common used metric system units and symbols are studied through manipulating the relationship between the units used in particular calculation. Moreover, to convert one metric system unit to another metric system, the first steps is by determining the relationship between the two system units. A pipette is a laboratory tool commonly used in biochemistry experiment to measured the volume of liquid, often as a media dispenser. Various type of pipette is investigated to different the levels of accuracy and precision. Pipette that dispense smaller volume are distinguished by micropipette whereas macropipette is used to dispense greater volumes. In this experiment, the Gilson pipetman(micropipette) and glass serological pipettes are used to transfer the volume of solution. During deliver the volume, the tip is place straight vertically into the receiving vessel and the plunger is press all the way towards the bottom of the test tube. This would help in expel or suck off all the liquid and gives a little extra volume to get the last droplet out. The calibration of the micropipette can be checked by using the fact of 1ml deionise(or distilled) water has a mass of 1g.The serological pipette frequently used in the laboratory as to transfer the millilitres volume of liquid from less than 1ml up to 50ml. The benefit of applying serological pipette is to mix chemical solutions or cell suspensions, and transfer the liquids between receptacles or layering the reagent of different densities carefully. While reading at the volume, be sure that the view of the pipette dead-on at eye level with the pipette held vertically, perpendicular to the ground. Volumes can only be measured when the entire volume is delivered and the pipette drained completely.
Dilution is a process that causing a substance to be less concentrated which usually simply by just adding a solvent such as water. Through this experiment, three type of calculation is been determined which included dilution factor, serial dilution and dilute using the V1C1=V2C2 method. A dilution factor is the ratio of final volume/ solute volume. This ways of expressing a dilution is without calculate any concentration of the solute. The dilution factor can be used as the denominator of the fraction such as DF of a 10 means a 1:10 dilution. More intermediary dilution is needed when a dilution factor is larger than the final volume needed or the amount of the stock is too small to be pipette. Besides that, the dilution can also be calculate by using the formula C1V1 = C2V2. This formula is used to produce a fixed amount of a dilute solution from a stock solution. A serial dilution is a repeating dilution of a substance in a solution as to achieve a geometric dilution of the original solution. It is widely used to create highly diluted solution accurately as well as solution for experiments which give a concentration curves with a logarithmic scale. The experiment result show that only test tube A (first dilution) shows the change of colour to brownish yellow. Other test tube shows similar test tube colour. This inaccurate result is due to the operative error done by us. One of it is different in eye level during transferring the volume of water sample and starch from one test tube to the other test tube. During the experiment there are several precautions during the experiment which included not shaking vertically so that the starch does not stain our thumb. In addition, make sure that the time for heating the solution does not overshoot 5 minutes as this will affect the final result of the result. In conclusion, we should be cautious in every step of the experiment as to minimize the error for the result. During the serial dilution, we should only let one personal to transfer the solution as to prevent or minimize the happen of different eye level which has high affinity to causes an inaccuracy result. We also had to put focus towards the temperature and time for heating the solution because there is the factor that affects the reaction.
CONCLUSION In this experiment, is aims to provide familiarity with basic biochemistry laboratory techniques to students who take biochemistry and to develop the ability to think scientifically and evaluate information critically . In addition, we had conducted an experiment on dilution in order to learn how to calculate dilution factor as well as calculating the starch concentration by doing serial dilutions. From this experiment, we also had learned how to handle the pipette using the proper ways, and hence successfully obtained the results.
REFERENCE 1. Cleveland, W. S. (1985). The elements of graphing data. Monterey, CA: Wadsworth 2. Kosslyn, S. M. (1994). Elements of graph design. San Francisco, CA: W. H. Freeman 3. JoVE Science Education Database. General Laboratory Techniques. Introduction to Serological Pipettes and Pipettors. JoVE, Cambridge, MA, doi: 10.3791/5034 (2014). 4. www.thefreedictionary.com 5. www.distichus.com
Exercise 2: Volume: 1 liter = 1000ml, 1ml = 1000 μl Please give detail calculation on each of the exercise given. 1.To calculate 0.15 liters = 150 000 μl. 0.15 x 1000 x 1000 =150 000 µl 2. To calculate 4310 μl = 4.31 x 10-3 liters. 4310÷1000÷1000 =4.31 x 10-3 3. To calculate 0.35 liters= 350 000 μl. 0.35 x 1000 x 1000 =350 000 µl
Weight: 1 gram = 1000mg 1 mg = 1000 μg 1 μg = 1000 ng 1 ng = 1000 pg 1. To calculate 36.2 mg = 3.62 x 1010 pg. 36.2 x 1000 x 1000 x 1000 = 3.62 x 1010 2. To calculate 67 μg = 6.7 x 10-5 g. 67 ÷ 1000 ÷ 1000 =6.7 x 10-5 3. To calculate 366 μg = 3.66 x 10-4 g. 366 ÷ 1000 ÷ 1000 =3.66 x 10 -4
Molarity: 1 mole = 1000 mmole, 1 mmole = 1 μmole The same logic applies to convert between the units. Sometimes, you will need to convert more than one unit. 1. To calculate 17 μg/ml = 0.017 g/liter µg
g
=17÷ 1000 ÷ 1000 =17 g/ml ml
liter
=17 ÷ 1000 = 0.017 g/liter 2. To calculate 60mg/ml = 60 g/liter mg
g
= 60 ÷ 1000 g/ml ml
liter
= 60 ÷ 1000 ÷ 1000 = 60 g/liter 3. To calculate 654 μg/ml = 0.654 g/liter µg
g
= 654 ÷ 1000 ÷ 1000 = 654 g/ml ml
liter
= 654 ÷ 1000 =0.654 g/liter
Dilution
Vol Concentrate
Vol Buffer
Final Vol
1:5
1 ml
4 ml
5 ml
1:5
1 ml
4 ml
5 ml
1:4
3 ml
9 ml
12 ml
1:2
1 ml
1 ml
2 ml
1:1000
10 ml
990 ml
1000 ml
1:2:5
1 ml
1.5 ml
2.5 ml
Exercise 3: 1. Please fill in the empty slots with appropriate value. 2. In the experiment below, you have a protein solution (a stock solution) at 2 mg/ml. You need to make the following levels: 400 μg /ml, 100 μg /ml, 20 μg /ml, 5 μg /ml and 1 μg /ml. You need 50 ml of each concentration.
Final Concentrati on
Calculate Dilution
Dilution
Vol stock
Vol buffer
Final Vol
400 μg /ml
M1V1=M2V2 2mg/ml=2000 μg /ml (2000)(x)=(400) (50) 2000 x=20 000 x= 10 μg /ml
10 : 50
10 ml
40 ml
50 ml
M1V1=M2V2 2mg/ml=2000 μg /ml (2000)(x)=(100) (50) 2000 x=5000 x= 2.5 μg /ml
2.5 : 50
2.5 ml
47.5 ml
65 ml
M1V1=M2V2 2mg/ml=2000 μg /ml (2000)(x)=(20)(65) 2000 x=1300
0.65 : 65
0.65 ml
64.35 ml
50 ml
100 μg /ml
20 μg /ml
1:5
1 : 20
x= 0.65 μg /ml 5 μg /ml
M1V1=M2V2 2mg/ml=2000 μg /ml (2000)(x)=(5)(50) 2000 x=250 x= 0.125 μg /ml
0.125 : 50
0.125 ml
49.875 ml
50 ml
1 μg /ml
M1V1=M2V2 2mg/ml=2000 μg /ml (2000)(x)=(1)(50) 2000 x=50 x= 0.025 μg /ml
0.025 : 50
0.025 ml
49.975 ml
50 ml
3. The 5 μg /ml and the 1 μg /ml solutions have large dilutions and volumes of the stock too small to pipet accurately. Sometimes it is helpful to use a second less concentrated stock solution. Suppose we made an extra quantity of the 20 μg /ml level and used that to make these solutions. How much extra beyond 50 ml do we need? Concentration
Calculate Dilution
Dilution
Vol stock
Vol buffer
Final Vol
5 μg /ml
V1C1 = V2C2
5 : 20
12.5 ml
37.5 ml
50 ml
(V1)(20) = (50)(5)
1:4
25 ml
25 ml
50 ml
20(V1) = 250 10 μg /ml
V1 = 12.5 ml V1C1 = V2C2 (V1)(20) = (50)(10)
10 : 20 1:2
20(V1)= 500 V1 = 25 ml We need an extra of the 37.5 ml of 20 µg/ml level, so the quantities for that concentration have been adjusted in the table above.
Exercise 4: Initial Vol solution Vol buffer Final Concentration Concentration 100 mg/ml 5 ml of 100 mg/ml 5 ml 50 mg/ml 50 mg/ml 5 ml of 50 mg/ml 25 mg/ml 25 mg/ml 5 ml of 25 mg/ml 12.5 mg/ml 12.5 mg/ml 5 ml of 12.5 mg/ml 6.25 mg/ml 6.25 mg/ml 5 ml of 6.25 mg/ml 3.125 mg/ml The advantage of this method is ease of pipeting. The disadvantage is that an error in one dilution causes errors for all the remaining dilutions. Calculation : V1C1 = V2C2 (5)(100) = (10)(x) 500 = 10x x = 50 V1C1 = V2C2 (5)(50) = (10)(x) 250 = 10x x= 25 V1C1 = V2C2 (5)(25) = (10)(x) 125 = 10x x=12.5 V1C1 = V2C2 (5)(12.5) = (10)(x) 62.5 = 10x x= 6.25 V1C1 = V2C2 (5)(6.25)= (10)(x) 31.25 = 10x x= 3.125
