1. The Basic Skills of the Biotechnology Workplace
Chapter 3

2. Learning Outcomes
Determine the most appropriate tool for measuring specific volumes or masses
Describe how to select, set, and use a variety of micropipets within their designated ranges to accurately measure small volumes
Convert between units of measure using the B <- -> S rule and appropriate conversion factors
Recognize the different expressions for units of concentration measurements and use their corresponding equations to calculate the amount of solute needed to make a specified solution
Define the term buffer and calculate a specified dilution from a concentrated stock solution or buffer

3. 3.1 Measuring Volumes in a Biotechnology Facility
Volume is a measurement of the amount of space something occupies
Volume is measured in:
Cubic centimeters (cc or cm3)
Liters (L)
Milliliters (mL)
Microliters (mL)

4. Metric Prefixes

5. Metric Prefixes To convert to a smaller unit, move
decimal point to the right or multiply.
Kilo -
1000
units k
Hecto -
100
units h
Deka - 10
units da
Basic
Unit
Deci -
0.1
units d
Centi -
0.01
units c
Milli -
0.001
units m
To convert to a larger unit, move
decimal point to the left or divide.
Micro-
Units m
Nano -
units n

6. Different tools are used to measure volume
pipet
Graduated cylinder
micropipette

7. Converting Units
Often volumes are measured in one unit of measurement and reported in another
Converting between metric units
Conversion factor
To measure volumes larger than 10 milliliters, technicians usually use a graduated cylinder

8. Reading a graduated cylinder
Reading a graduated cylinder. Before using a graduated cylinder, make sure you know the total volume it will hold and the value of each of the graduations. In the lab, common graduated cylinders include 10 mL, 25 mL, 100 mL, 250 mL, 500 mL, and 1 L.
Pipets are available that measure volumes between 0.1 mL and 50 mL. Shown from left to right are 25-, 10-, 5-, and 1-mL pipets.

9. Picking and Using the Appropriate Micropipet
Using Pipets
Picking and Using the Appropriate Micropipet
Measuring units smaller than 10 mL requires a pipet.
“Never mouth pipet!”
P-100 or P-200 micropipet
P-10 or P-20 micropipet
P-1000 micropipet
Using Micropipets
When measuring tiny volumes, less that 1 mL, a micropipet is used.
A micropipet has four parts
Plunger button
Ejector button
Volume display
Dispensing tip

10. 200
- 20
Labeled micropipet. Learning to use each part of a micropipet correctly is essential. On the micropipet shown, the plunger has two “stops.” Pressing to the first stop evacuates air to the volume in the display. Pressing to the second stop evacuates that volume plus another 50% or so. To ensure accurate measurement, feel the difference between the first and second stop before using the pipet. Inaccurate measurement could waste costly reagents and cause invalid experiment results.
P-200
P-100 Micropipet. This micropipet will measure volumes as small as 20 mL.

11. 20
- 2
P - 20
P-10 Micropipet. This pipet measures volumes as small as 2.0 mL. A P-20 uses tiny tips that are usually white.
P-1000 Micropipet. A P-1000 micropipet will measure up to 1000 mL, or 1 mL, and uses large tips that are usually blue or white in color.

12. A multichannel pipet allows several samples to be measured at the same time, a feature that saves time during an experiment with multiple replications and repetitive pipeting.

13. What instrument would you use to measure and dispense 23.5 µL
2-20 µL / P-20 micropipet
µL / P-200 micropipet
µL / P-1000 micropipet
Pipet: 10 ml / 5 ml / 2 ml / 1 ml
Graduated cylinder

14. What instrument would you use to measure and dispense 6.5 mL
2-20 µL / P-20 micropipet
µL / P-200 micropipet
µL / P-1000 micropipet
Pipet: 10 ml / 5 ml / 2 ml / 1 ml
Graduated cylinder

15. What instrument would you use to measure and dispense 125 mL
2-20 µL / P-20 micropipet
µL / P-200 micropipet
µL / P-1000 micropipet
Pipet: 10 ml / 5 ml / 2 ml / 1 ml
Graduated cylinder

16. What instrument would you use to measure and dispense 7 L
2-20 µL / P-20 micropipet
µL / P-200 micropipet
µL / P-1000 micropipet
Pipet: 10 ml / 5 ml / 2 ml / 1 ml
Graduated cylinder

17. What instrument would you use to measure and dispense 2.87 mL
2-20 µL / P-20 micropipet
µL / P-200 micropipet
µL / P-1000 micropipet
Pipet: 10 ml / 5 ml / 2 ml / 1 ml
Graduated cylinder

18. What instrument would you use to measure and dispense 555 L
mL micropipette
2-20 mL micropipette
mL micropipette
Pipette
Graduated cylinder

19. Convert the following units to the requested unit:
1.7 L = 1700 mL
235.1 µL = mL
2.37mL = 2370 µL
What numbers should be dialed into the P-20 display if a volume of 3.7 µL is to be measured? 03 7

20. What instrument should be used if a technician wants to fill 40 sets of 16 tubes all with identical volumes?
mL micropipette
2-20 mL micropipette
Multichannel micropipette
mL micropipette
Pipette
Graduated cylinder

21. 3.2 Solutions
Solution preparation is a basic, essential skill of a biotechnology lab employee.
Aqueous solution – proteins or nucleic acids in a watery solution
Solutions are a mixture of two or more substances where one (solute) completely dissolves in the other (solvent)

22. Solutes Solute – the substance in a solution that is being dissolved
– the substance in the lesser amount in the solution
Solid solutes are measured on balances or scales.

23. Tabletop Balance: ours measures to 0.01g

24. Analytical balances - measure down to milligrams, even though they usually report in grams.

25. Solvents Solvent – the substance that dissolves the solute
- the substance in the greater amount in the solution
Liquid solvents are measured in graduated cylinders, pipets, or micropipets

26. Always prepare in clean containers to avoid contamination of solutions that interfere with reactions:
Wash with lab soap and water
Rinse with tap water until no evidence of soap
Rinse 5 X with tap water
Final rinse with deionized water if available

27. Solution Concentration: portion of solute to solvent
Concentration is measured in several ways:
Mass/volume (ex. g/L, mg/ml, µg/ml, µg/µL)
Volume/volume
% mass/volume
Molarity (ex. M, mM, µM)
Normality (acids & bases only)

28. Solution Concentration
Molarity –concentration that represents the number of moles of a solute in a liter of solution (or some fraction of that unit)
Normality –concentration generally used for acids and bases that is expressed in gram equivalent weights of solute per liter of solution; represents the amount of ionization of an acid or base

29. What instrument should be used to measure and dispense the following solutes?
_____3.5 g of salt _____6.5 mg of DNA _____2.5 g of gelatin
Electronic Tabletop Balance
Electronic Analytical Balance

30. What is the relation of solute to solvent as a solution becomes more concentrated?
The amount of solute increases in relation to the amount of solvent as a solution becomes more concentrated.

31. Which of the following are concentration units? mi/hr g/mL mM °F/°C
g/mL and mM

32. 3.3 Solutions of Given Mass/Volume Concentrations
Mass/Volume Solution. Solvent is added until a volume of 10 mL is reached. A protein solution that has a concentration of 1 mg/mL is considered fairly concentrated.

33. Making Mass/Volume Solutions
Concentration in Mass/Volume Units
____ g/mL X ____ mL = ____ g of solute
concentration volume to be weighed out,
desired desired dissolved in the solvent

34. Practice Problems: mass/vol concentration
How many grams of hemoglobin are needed to make 100 ml of a 0.05 g/ml hemoglobin solution?
0.05 g X 100 ml = _____ g of hemoglobin ml
Answer: 5 g

35. Practice Problems: mass/vol concentration
How many grams of hemoglobin are needed to make 150 ml of a 1 mg/ml hemoglobin solution?
1 mg X 150 ml = _____ mg of hemoglobin ml
Answer: 150 mg – Convert to grams for massing:
150 mg = g

36. Practice Problems: mass/vol concentration
How many grams of hemoglobin are needed to make 100 ml of a 50 µg/ml hemoglobin solution?
50 _µg X 100 ml = _____ µg of hemoglobin ml
Answer: µg – Convert to grams for massing:
5000 µg = g

37. Which of the following are mass/volume concentration units?
mg/mL
g/mg
L/mg
D. mg/mL
g/l

38. How many grams of the protein, gelatin, are needed to make 0
How many grams of the protein, gelatin, are needed to make 0.5 L of a 3 g/L gelatin solution?
Set-up the equation and solve:
The correct answer is 1.5 g

39. How many grams of sugar are need to make 25 mL of a 25 mg/mL sugar solution?
Set-up and solve:
The correct answer is: 625 mg = _____g or .625 g

40. How many grams of salt are needed to make 150 mL of a 100 mg/mL salt solution?
Set-up and solve:
The correct answer is µg = _____g
or g

41. 3.4 Solutions of Differing % Mass/Volume Concentrations
A percentage represents something that is part of 100.
What is the decimal equivalent of the following percentages?
10% - 10% of 100 = 10 , 10/100 = .10
15%
25% - .25 2%
1.5%
0.5%

42. Using decimal equivalents solve the following:
50% of 10 = .5 X 10 = 5
50% of 1 = .5 X 1 = .5
20% of 40 = .2 X 40 = 8
10% of 40 = .1 X 40 = 4
5% of 40 = .05 X 40 = 2

43. Solutions of Differing % Mass/Volume Concentrations
Concentration % Mass/Volume Equation
____ % = ____
percent value decimal value of the g/mL
____ X ____ = ____ g of solute to
decimal total volume be measured and
value desired (mL) added to the volume desired of solvent

44. What mass of gelatin (a protein) is needed to make 0
What mass of gelatin (a protein) is needed to make 0.5 L of a 3% gelatin solution?
____ X ____ = ____ g of solute to decimal total volume be measured and value desired (mL) added to the volume desired of solvent
.03 g/ml X 500 ml = 15 g

45. What mass of sugar is needed to make 25 mL of a 2.5% sugar solution?
____ X ____ = ____ g of solute to decimal total volume be measured and value desired (mL) added to the volume desired of solvent
.025 g/ml X 25 ml = g

46. What mass of salt is needed to make 150 mL of a 10% salt solution?
____ X ____ = ____ g of solute to decimal total volume be measured and value desired (mL) added to the volume desired of solvent
.1 g/ml X 150 ml = 15 g

47. Molar Concentrations…
Mole – the mass, in grams, of 6 X 1023 atoms of molecules of a given substance; one mole is equivalent to the molecular weight of a given substance, reported in grams

48. Molar Concentrations…
Molecular weight – the sum of all the atomic weights of the atoms in a given molecule
amu – abbreviation of atomic mass unit; the mass of a single hydrogen atom

49. To find molecular weight:
Determine what atoms are in a compound and how many of each are present
C6H12O6 contains
6 atoms C;
12 atoms H
6 atoms O

50. Continuing… Each Carbon atom has a weight of 12 amu
Each Hydrogen atom has a weight of 1 amu
Each Oxygen atom has a weight of 16 amu
How do I know these weights?? I look on the periodic table! (World’s Biggest Cheat Sheet!!)

51. Even more about molecular weight…
C6H12O6
6 atoms C X 12 amu/atom = 72 amu
12 atoms H X 1 amu/atom = 12 amu
6 atoms O X 16 amu/atom = 96 amu
Total = amu total weight of glucose

52. 3.5 Solutions of Differing Molar Concentrations
Molarity Concentration Equation
volume molarity molecular the number of grams to be
wanted (L) X desired X weight of the = dissolved in solvent, up to
(mol/L) solute (g/mol) the total volume of
solution desired

53. Periodic Table. The Period Table of Elements shows the elements (atoms) found in compounds (molecules). Each element is listed along with the atomic weight (mass) of each atom in the element. A NaCl molecule has a molecular weight of about 58.4 atu (atomic mass units) because the Na atom weighs about 23 amu, and the Cl atom weighs about 35.4 amu. Together, in the NaCl molecule, the atoms total approximately 58.4 amu. The mass of a hydrogen atom equals 1 amu.

54. mass spectrometer - Scientists use it to determine the molecular weight of a compound. A “mass spec” can also determine if a sample is contaminated with molecules of different molecular weights.

55. What is the molecular weight of each of the following compounds?
NaOH:
Na = 23
O = 16
H = 1
40 amu

56. MgCl2:
Mg = 24
Cl = 35.5 X2 = 71
95 amu

57. MgO:
Mg = 24
O = 16
40 amu

58. HCl:
H = 1
CL = 35.5
36.5 amu

59. NaCl:
Na = 23
CL = 35.5
58.5 amu

60. What mass of NaCl is needed for 0.5 L of a 0.5 M NaCl solution?
volume molarity molecular the number of grams to be wanted (L) X desired X weight of the = dissolved in solvent, up to (mol/L) solute (g/mol) the total volume of solution desired
Na = 23
Cl = 35.5
58.5
.5L X .5 M X 58.5 g = g NaCl
L M
14.65 g brought to a final volume of 500 mL

61. What mass of MgO is needed for 200 mL of a .025 M MgO solution?
volume molarity molecular the number of grams to be wanted (L) X desired X weight of the = dissolved in solvent, up to (mol/L) solute (g/mol) the total volume of solution desired
MgO:
Mg = 24
O = 16 40
.2 L X M X 40 g = g MgO
L M
Brought to a final volume of .2 L or 200 mL

62. What mass of sodium hydroxide (NaOH) is needed to make 750 mL of a 125 mM NaOH solution?
volume molarity molecular the number of grams to be wanted (L) X desired X weight of the = dissolved in solvent, up to (mol/L) solute (g/mol) the total volume of solution desired
NaOH:
Na = 23
O = 16
H = 1 40
.75 L X M X g = g of NaOH
L M
Brought to a final volume of 750 mL

63. 3.6 Dilutions of Concentrated Solutions
Reason for concentrated stock solutions:
-easier to prepare higher conc. solutions (tiny amts. difficult to mass.
Concentrating 1 L Solution. Many chemical and biological reagents are purchased in concentrated form. Concentrated solutions can be prepared initially with a greater amount of solute to solvent, or a solution can be concentrated by removing water. A diluted solution can be prepared by adding solvent to the solution.

64. Dilution – the process in which solvent is added to make a solution less concentrated
Diluting a 100 mg/mL Stock Solution to 1 mg/mL.

65. To Calculate Dilutions…
C1V1 = C2V2
C1 = original stock concentration
V1 = amount of stock solution to use
C2 = new more dilute concentration
V2 = desired volume of dilute solution

66. V1 = the volume to use of the stock soln C2 = 1 mg/mL V2 = 1L
Ex. How would you make 1L of 1 mg/mL protein solution from 100 mg/mL concentrated stock solution?
C1 = 100 mg/mL
V1 = the volume to use of the stock soln
C2 = 1 mg/mL
V2 = 1L
C1V1 = C2V2
100 mg/mL X _____= 1 mg/mL X 1000ml
100 X V1 = 1000 mg
V1 = 1000mg / 100 mg/mL
V1 = 10 mL

67. How do you make 200 mL of 75 µM CaCl2 solution from 10 mM CaCl2 solution?

68. Terms:
Buffers – used to store proteins and nucleic acids
- solution that acts to resist a change in pH when the hydrogen ion concentration is changed.
-depending on the kind of buffer needed, different salts at various concentrations are used
TRIS – common buffer salt which is a complex organic molecule used to maintain the pH of a soln.
TE Buffer – buffer used for storing DNA; contains TRIS and EDTA
TAE buffer – used for running DNA samples on agarose gels in horizontal gel boxes; contains TRIS, EDTA, and acetic acid.

69. How can we make 600 ml of 1X TAE buffer from 50X stock solution?
C1V1 = C2V2
C1 = 50X V1 = ???
C2 = 1X V2 = 600ml
50x * V1 = 1X * 600 ml
50x * V1 = 600 Xml
50X X
V1 = 12 ml to make 600… ml of water

70. How do you prepare 40 mL of a 2 mg/mL protein solution from 10 mg/mL protein solution?

71. How do you prepare 200 mL of 2X enzyme buffer from 10X enzyme buffer solution?

72. How do you prepare 500 mL of 50 mM NaCl solution from 5 mM NaCl solution?

73. How do you prepare 3 L of 1X TAE buffer from 50X TAE buffer stock solution?