Biotech Chapter Three Lecture- Basic Lab Skills

The Basic Skills of the Biotechnology Workplace

Chapter 3

Learning Outcomes

Determine the most appropriate tool for measuring specific volumes of masses

Learning Outcomes


Describe how to select, set, and use a variety of micropipets within their designated ranges to accurately measure small volumes

Learning Outcomes


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

Learning Outcomes

Learning Outcomes

Learning Outcomes

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

Learning Outcomes

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

Describe what pH is and why it is important in solution preparation

3.1 Measuring Volumes in a Biotechnology Facility


Volume is a measurement of the amount of space something occupies





Volume is measured in• Liters (L)• Milliliters (mL) • Microliters (µL)



Volume is measured in• Liters (L)• Milliliters (mL) • Microliters (µL)

Different tools are used to measure volume• Graduated cylinder• Pipet• Micropipet

Converting Units

Often volumes are measured in one unit of measurement and reported in another

Converting Units


•Converting between metric units

Converting Units



move decimal point

Converting Units



move decimal point

B ↔ S

Converting Units



move decimal point

B ↔ S

•Use a conversion factor for non-metric

To measure volumes larger than 10 milliliters, technicians

usually use 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.

A volumetric flask is a more precise choice

will only measure one quantity

25-, 10-, 5-, and 1-mL SEROLOGICAL

pipets= pretty accurate

Using Pipets•Measuring units smaller

than 10 mL requires a pipet.

• “Never mouth pipet!”

VOLUMETRIC pipets

= better accuracy

Using Micropipets

When measuring tiny volumes, less that 1 mL, a micropipet is used.

Picking and Using the Appropriate Micropipet

•P-100 or P-200 micropipet•P-10 or P-20 micropipet•P-1000 micropipet

A micropipet has four parts• Plunger button• Ejector button• Volume display• Dispensing tip

Learning to use each part of a micropipet correctly is essential.

Inaccurate measurement could waste costly reagents and cause

invalid experiment results.

On the micropipet shown, the plunger has two “stops.”


Pressing to the first stop evacuates air to the volume in the display. Used for

drawing liquid.



drawing liquid.Pressing to the second stop evacuates

that volume plus another 50% or so. Used to dispense.



drawing liquid.Pressing to the second stop evacuates

that volume plus another 50% or so. Used to dispense.

To ensure accurate measurement, feel the difference between the first and second

stop before using the pipet.

P-1000 Micropipet. 100- 1000 µL (1 mL)

large tips are usually blue or white in color.

P-100 Micropipet. 10 - 100 µL

precision to 0.2 µLyellow tips

P-10 Micropipet.P-10 micropipets are

common in biotechnology labs.

0.5 - 10.0 µL precision to 0.02 µL.

tiny tips are usually white

Picking and Using the Appropriate Micropipet

Always use the smallest device possible.

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.

Vocabulary

Vocabulary

• Volume – a measurement of the amount of space something occupies

Vocabulary


• Mass – the amount of matter (atoms and molecules) an object contains

Vocabulary



• Liter – abbreviated “L”; a unit of measure for volume, approximately equal to a quart

Vocabulary




• Milliliter – abbreviated “mL”; a unit of measure for volume; one one-thousandth of a liter (0.001 L) or about equal to one-half teaspoon

Vocabulary





• Microliter – abbreviate “µL”; a unit of measure for volume; equivalent to one-thousandth of a milliliter or about the size of the tiniest teardrop

Vocabulary





• Microliter – abbreviate “µL”; a unit of measure for volume; equivalent to one-thousandth of a milliliter or about the size of the tiniest teardrop

• Graduated cylinder – a plastic tube with marks (or graduations) equally spaced to show volumes; measurements are made at the bottom of the meniscus, the lowest part of the concave surface of the liquid in the cylinder

Vocabulary

Vocabulary

• Pipet – an instrument usually used to measure volumes between 0.1 mL and 50 mL

Vocabulary


• Micropipet – an instrument used to measure very tiny volumes, usually less than a milliliter

Vocabulary



• Unit of measurement – the form in which something is measured (g, mg, µg, L, mL, µL, km, cm, etc.)

Vocabulary




• Conversion factor – a number (a fraction) where the numerator and denominator are equal to the same amount; commonly used to convert from one unit to another

Vocabulary




• Conversion factor – a number (a fraction) where the numerator and denominator are equal to the same amount; commonly used to convert from one unit to another

• Metrics conversion table – a chart that shows how one unit of measure relates to another (for example, how many milliliters are in a liter)

3.1 Review Questions

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1. What instrument would you use to measure and dispense the following volumes? Pick the instrument that is likely to give you the least error for each measurement.

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23.5µL 6.5mL 125mL 7µL 2.87mL 555µL

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23.5µL 6.5mL 125mL 7µL 2.87mL 555µL

2. Convert the following units to the requested unit:

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23.5µL 6.5mL 125mL 7µL 2.87mL 555µL


1.7 L = _____ mL 235.1 µL = _____ mL

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23.5µL 6.5mL 125mL 7µL 2.87mL 555µL


1.7 L = _____ mL 235.1 µL = _____ mL

2.37 mL = _____ µL

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23.5µL 6.5mL 125mL 7µL 2.87mL 555µL


1.7 L = _____ mL 235.1 µL = _____ mL

2.37 mL = _____ µL

3. What numbers should be dialed into a P-10 display if a volume of 3.7 µL is to be measured? 0 0 0



23.5µL 6.5mL 125mL 7µL 2.87mL 555µL


1.7 L = _____ mL 235.1 µL = _____ mL

2.37 mL = _____ µL

3. What numbers should be dialed into a P-10 display if a volume of 3.7 µL is to be measured?

4. What instrument should be used if a technician wants to fill 40 sets of 16 tubes all with identical volumes?

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3.2 Making Solutions•Solution preparation is one of the most essential skills of a biotechnology lab employee.


•Solutions are mixtures in which one or more substances are dissolved in another substance.



•Solid solutes are measured on balances or scales.



•Solid solutes are measured on balances or scales.

•Concentration is measured in several ways:•Mass/volume ●Volume/volume•% mass or % volume ●Molarity•Normality

3.2 Making SolutionsTo make any solution-1) calculate quantity of solute needed for desired amount of solution. amount of solution x concentration = amount of solute

(if units agree)


(if units agree)

2) Measure amount of solute


(if units agree)


3) Add solvent to desired volume


(if units agree)



4) All measuring devices must be clean (rinsed with deionized water) and completely dry OR rinsed with the material to be measured.


(if units agree)



4) All measuring devices must be clean (rinsed with deionized water) and completely dry OR rinsed with the material to be measured. Dirty glassware will contaminate/increase solute.Wet glassware will dilute the solution.

3.3 Mass/Volume Solutions

Concentration is measured in several ways:Mass/volume


•Do concentration calculations to determine the amount of solute required to produce a certain volume.Ex. 200ml of a 5g/L solutionrequires 5g/L x .2L = 1g solute




•Measure desired mass of solute using a balance




•Measure desired mass of solute using a balance

•Add solvent to desired volume


Mass/Volume Solution. Solvent is added to 10g protein until a volume of 10 mL is reached. A protein solution that has a

concentration of 1 g/mL is considered fairly concentrated.

An electronic balance is good for measuring 1-200

grams.

Most analytical balances measure

down to milligrams, even though they usually report in

grams.

3.2 Review Questions1. What instrument should be used to measure and

dispense the following solutes? 3.5 g of salt

6.5 mg of DNA 12.500 g of gelatin




2. What happens to the ratio of solute molecules to solvent as a solution becomes more concentrated?





3. Which of the following are concentration units?

mi/hr g/mL mM °F/°C





3. Which of the following are concentration units?

mi/hr g/mL mM °F/°C

4. Describe how glassware should be prepared before using it to prepare or store solutions.

1. Which of the following are mass/volume concentration units?

mg/mL g/mg L/mg µg/µL g/l




2. What mass of the protein, gelatin, is needed to make 0.5 L of a 3 g/L gelatin solution?





3. What mass of sugar is need to make 25 mL of a 25 mg/mL sugar solution?





3. What mass of sugar is need to make 25 mL of a 25 mg/mL sugar solution?

4. What mass of salt is needed to make 150 mL of a 100 µg/mL salt solution? Describe how the solution is prepared.


3.4- v/v and w/w solutions

Concentration is measured in several ways:Volume/volume


1) calculate solute amount2 liters of a 20ml/L solution requires 40ml of solute (2L x 20ml/L = 40ml)




2) Measure solute with cylinder or pipet.




2) Measure solute with cylinder or pipet.

3) Add solvent to final volume (1960ml)


Concentration is measured in several ways:

% mass or % volume



% mass or % volume

A %(v/v) solution is made the same as a volume/volume solution, but concentration is expressed as a percent.



% mass or % volume


A 20ml/L solution is 20ml/1000ml, which is 0.02 or 2%(v/v).



% mass or % volume


A 20ml/L solution is 20ml/1000ml, which is 0.02 or 2%(v/v).

A %(w/w) solution is very similar but solute & solvent must be weighed.


1. What is the decimal equivalent of the following percentages?

10% 15% 25% 2% 1.5% 0.5%



10% 15% 25% 2% 1.5% 0.5%

2. What mass of gelatin (a protein) is needed to make 0.5 L of a 3% gelatin solution?



10% 15% 25% 2% 1.5% 0.5%


3. What mass of sugar is needed to make 25 mL of a 2.5% sugar solution?



10% 15% 25% 2% 1.5% 0.5%


3. What mass of sugar is needed to make 25 mL of a 2.5% sugar solution?

4. What mass of salt is needed to make 150 mL of a 10% salt solution? Describe how the solution is prepared.


3.5 MolarityConcentration is measured in several ways:Molarity = moles/Liter

Concentration is measured in several ways:Molarity = moles/Liter1) Determine moles of solute required.

Molarity x volume of solution = moles solute 250ml of 2M NaCl needs

0.25L x 2M = 0.5moles NaCl

3.5 Molarity



0.25L x 2M = 0.5moles NaCl2) Convert moles to grams (multiply by molar mass of solute)

0.5mol NaCl x 58.5g/mol = 29.25g NaCl

3.5 Molarity




0.5mol NaCl x 58.5g/mol = 29.25g NaCl3) Mass required amount of solute.

3.5 Molarity




0.5mol NaCl x 58.5g/mol = 29.25g NaCl3) Mass required amount of solute.4) Add solvent to final volume.

3.5 Molarity

1. What is the molecular weight of each of the following compounds?

NaOH MgCl2 Mg(OH)2 HCl




2. What mass of NaCl is needed for 0.5 L of a 0.5 M NaCl solution?





3. What mass of MgCl2 is needed for 200 mL of a 0.025 M MgCl2 solution?





3. What mass of MgCl2 is needed for 200 mL of a .025 M MgCl2 solution?

4. What mass of sodium hydroxide (NaOH) is needed to make 750 mL of a 125 mM NaOH solution? Describe how to prepare the solution.


Vocabulary

Solution – a mixture of two or more substances where one (solute) completely dissolves in the other (solvent)

Vocabulary


Aqueous – describing a solution in which the solvent is water

Vocabulary



Solute – the substance in a solution that is being dissolved

Vocabulary




Balance – an instrument that measures mass

Vocabulary




Balance – an instrument that measures mass

Weight – the force exerted on something by gravity; at sea level, it is considered equal to the mass of an object

Vocabulary

Gram – abbreviated “g”; the standard unit of mass, approximately equal to the mass of a small paper clip

Vocabulary


Solvent – the substance that dissolves the solute

Vocabulary



Molarity – a measure of concentration that represents the number of moles of a solute in a liter of solution (or some fraction of that unit)

Vocabulary



Molarity – a measure of concentration that represents the number of moles of a solute in a liter of solution (or some fraction of that unit)

Normality – a measurement of concentration generally used for acids and bases that represents the amount of ionization of an acid or base. 2M HCl = 2N HCl 2M H2SO4 = 4N H2SO4

Percentage – a proportion of something out of 100 parts, expressed as a whole number

20/100 = 0.2 = 20%

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

Molecular weight – the sum of all the atomic weights of the atoms in a given molecule

Mass spectrometer – an instrument that is used to determine the molecular weight of a compound

Vocabulary

This instrument is a 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.

3.6 Dilutes of Concentrated Solutions

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.




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 a concentrated one.


Concentrationstock x Volumestock =

Concentrationdiluted x Volumediluted

CsVs = CdVd

Solve for the volume of stock solution needed

Diluting a 100 mg/mL Stock Solution to make 1L of 1 mg/mL.

100 mg/mL x Vs = 1 mg/mL x 1000mLneed 10 ml of stock

Dilution – the process in which solvent is added to make a solution less concentrated

Vocabulary


Stock solution – a concentrated form of a reagent that is often diluted to form a “working solution”

Vocabulary



Buffer – a solution that acts to resist a change in pH when the hydrogen ion concentration is changed

Vocabulary



Buffer – a solution that acts to resist a change in pH when the hydrogen ion concentration is changed

TRIS – a complex organic molecule used to maintain the pH of a solution

Vocabulary


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



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




3. How do you prepare 500 µL of 10 µM NaCl solution from 5 µM NaCl solution?




3. How do you prepare 500 µL of 10 µM NaCl solution from 5 µM NaCl solution?

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

Education

Biotech Chapter Three Lecture- Basic Lab Skills