Molecular Biology Technical Skills. Skills Micropipetting Preparing solutions Working with concentrations Dilutions Amounts Agarose gel electrophoresis

Molecular Biology

Technical SkillsTechnical Skills

SkillsSkills

Micropipetting Micropipetting Preparing solutionsPreparing solutions Working with concentrationsWorking with concentrations DilutionsDilutions AmountsAmounts Agarose gel electrophoresis Agarose gel electrophoresis

Micropipetting- Measuring Micropipetting- Measuring small volumessmall volumes

Allows to measure microliters (µL)Allows to measure microliters (µL) 1 000 X less than 1 milliliter1 000 X less than 1 milliliter

2-20 µL 50-200 µL 100-1000 µLMax. 0.02 mL 0.2mL 1mL

Setting the volume- P20Setting the volume- P20

Tens (0, 1=10 or 2=20)

Units (0-9)

Decimal (1-9 = 0.1-0.9)


Hundreds (0, 1=100 or 2=200)

Tens (0, 1-9=10-90)

Units (1-9)


Thousands (0, 1=1000)

Hundreds (0, 1-9=100-900)

Tens (0, 1-9=10 - 90)

Using the micropipettorUsing the micropipettor

Step 1Insert tip

Step 2Press plunger up to first stop

Step 3Insert tip in solution to be drawn

Step 4Draw up sample by slowly releasing plunger

Step 5Withdraw pipettor

Dispensing

Start dispensing 1st stop =Dispense 2nd stop = Expel

Guidelines for optimal Guidelines for optimal reproducibilityreproducibility

Use pipettor whose volume is closest to Use pipettor whose volume is closest to the one desiredthe one desired

Consistent SPEED and SMOOTHNESS to Consistent SPEED and SMOOTHNESS to press and release the PLUNGERpress and release the PLUNGER

Consistent IMMERSION DEPTH Consistent IMMERSION DEPTH 3-4mm below surface3-4mm below surface

AVOID air bubblesAVOID air bubbles NEVER go beyond the limits of the NEVER go beyond the limits of the

pipettorpipettor

Preparing Preparing SolutionsSolutions

DefinitionsDefinitions

SolutionSolution Mixture of 2 or more substances in a Mixture of 2 or more substances in a

single phasesingle phase Solutions are composed of two Solutions are composed of two

constituentsconstituents SoluteSolute

Part that is being dissolved or diluted – Usually Part that is being dissolved or diluted – Usually smaller amountsmaller amount

Solvent (OR Diluent)Solvent (OR Diluent)Part of solution in which solute is dissolved – Usually Part of solution in which solute is dissolved – Usually

greater volumegreater volume

Concentrations Concentration = Quantity of solute

Quantity of solution (Not solvent)

Three basic ways to express concentrations: Molar concentration (Molarity) Percentages Mass per volume

Molarity

# of Moles of solute/Liter of solution

Mass of solute/MW of solute = Moles of solute

Moles of solute/vol. in L of solution =

Molarity

PercentagesPercentages

Percentage concentrations can be Percentage concentrations can be expressed as either:expressed as either: V/V – volume of solute/100 mL of V/V – volume of solute/100 mL of

solutionsolution W/W – weight of solute/100g of solutionW/W – weight of solute/100g of solution W/V – Weight of solute/100mL of W/V – Weight of solute/100mL of

solutionsolution All represent fractions of All represent fractions of 100100

Percentages Percentages (Cont’d)(Cont’d)

%V/V%V/V Ex. 4.1L solute/55L solution =7.5%Ex. 4.1L solute/55L solution =7.5%

Must have same units top and bottom!Must have same units top and bottom!

%W/V%W/V Ex. 16g solute/50mL solution =32%Ex. 16g solute/50mL solution =32%

Must have units of same order of Must have units of same order of magnitude top and bottom!magnitude top and bottom!

% W/W% W/W Ex. 1.7g solute/35g solution =4.9%Ex. 1.7g solute/35g solution =4.9%

Must have same units top and bottom!Must have same units top and bottom!

DilutionsDilutions

Reducing a Reducing a ConcentrationConcentration

A FractionA Fraction

DilutionsDilutions

Dilution = making weaker Dilution = making weaker solutions from stronger onessolutions from stronger ones

Example: Making orange juice Example: Making orange juice from frozen concentrate. You mix from frozen concentrate. You mix one can of frozen orange juice with one can of frozen orange juice with three (3) cans of water.three (3) cans of water.

Dilutions (cont’d)Dilutions (cont’d)

Dilutions are expressed as the volume Dilutions are expressed as the volume of the solution being diluted per the of the solution being diluted per the total final volume of the dilutiontotal final volume of the dilution

In the orange juice example, the In the orange juice example, the dilution would be expressed as 1/4, for dilution would be expressed as 1/4, for one can of O.J. to a TOTAL of four cans one can of O.J. to a TOTAL of four cans of diluted O.J. When saying the of diluted O.J. When saying the dilution, you would say, in the O.J. dilution, you would say, in the O.J. example: “one in four”.example: “one in four”.


Another example:Another example: If you dilute 1 ml of serum with 9 If you dilute 1 ml of serum with 9

ml of saline, the dilution would be ml of saline, the dilution would be written 1/10 or said “one in ten”, written 1/10 or said “one in ten”, because you express the volume of because you express the volume of the solution being diluted (1 ml of the solution being diluted (1 ml of serum) per the TOTAL final volume serum) per the TOTAL final volume of the dilution (10 ml total).of the dilution (10 ml total).


Another example:Another example: One (1) part of concentrated acid One (1) part of concentrated acid

is diluted with 100 parts of water. is diluted with 100 parts of water. The total solution volume is 101 The total solution volume is 101 parts (1 part acid + 100 parts parts (1 part acid + 100 parts water). The dilution is written as water). The dilution is written as 1/101 or said “one in one hundred 1/101 or said “one in one hundred and one”.and one”.


Notice that dilutions do NOT have Notice that dilutions do NOT have units (cans, ml, or parts) but are units (cans, ml, or parts) but are expressed as one number to expressed as one number to another numberanother number Example: 1/10 or “one in ten”Example: 1/10 or “one in ten”


Dilutions are always expressed Dilutions are always expressed with the original substance diluted with the original substance diluted as one (1). If more than one part as one (1). If more than one part of original substance is initially of original substance is initially used, it is necessary to convert the used, it is necessary to convert the original substance part to one (1) original substance part to one (1) when the dilution is expressed.when the dilution is expressed.


Example:Example:Two (2) parts of dye are diluted with eight (8) parts of Two (2) parts of dye are diluted with eight (8) parts of diluent (the term used for the diluting solution). The diluent (the term used for the diluting solution). The total solution volume is 10 parts (2 parts dye + 8 total solution volume is 10 parts (2 parts dye + 8 parts diluent). The dilution is initially expressed as parts diluent). The dilution is initially expressed as 2/10, but the original substance must be expressed 2/10, but the original substance must be expressed as one (1). To get the original volume to one (1), use as one (1). To get the original volume to one (1), use a ratio and proportion equation, remembering that a ratio and proportion equation, remembering that dilutions are stated in terms of 1 to something:dilutions are stated in terms of 1 to something:____________2 parts dye 2 parts dye = ___ = ___1.0___1.0___ 10 parts total volume x10 parts total volume x

2 x = 102 x = 10 x = 5x = 5The dilution is expressed as 1/5.The dilution is expressed as 1/5.


The dilution does not always end up in whole numbers.The dilution does not always end up in whole numbers.Example:Example:Two parts (2) parts of whole blood are diluted with Two parts (2) parts of whole blood are diluted with five (5) parts of saline. The total solution volume is five (5) parts of saline. The total solution volume is seven (7) parts (2 parts of whole blood + 5 parts seven (7) parts (2 parts of whole blood + 5 parts saline). The dilution would be 2/7, or, more correctly, saline). The dilution would be 2/7, or, more correctly, 1/3.5. Again, this is calculated by using the ratio and 1/3.5. Again, this is calculated by using the ratio and proportion equation, remembering that dilutions are proportion equation, remembering that dilutions are stated in terms of 1 to something: stated in terms of 1 to something:

____2 parts blood_____2 parts blood_____ = ___ = ___1.0___1.0___ 7 parts total volume x7 parts total volume x

2 x = 72 x = 7 x = 3.5x = 3.5

The dilution is expressed as 1/3.5The dilution is expressed as 1/3.5

What Does This Mean??What Does This Mean??

If a solution has a 1/10 dilution the If a solution has a 1/10 dilution the fraction represents 1 part of the fraction represents 1 part of the sample being diluted added to 9 sample being diluted added to 9 parts of diluent for a total of 10 parts of diluent for a total of 10 parts. parts.

If this solution was prepared to a If this solution was prepared to a final volume of 110 mL, what final volume of 110 mL, what volumes of solute and what volume volumes of solute and what volume of solvent have to be used?of solvent have to be used?

In other words, what is the volume of In other words, what is the volume of 1 part and of 9 parts?1 part and of 9 parts?

Dilution FactorDilution Factor EXAMPLE: What is the dilution factor if EXAMPLE: What is the dilution factor if

you add 0.1 mL aliquot of a specimen to you add 0.1 mL aliquot of a specimen to 9.9 mL of diluent?9.9 mL of diluent? The final volume is equal to the aliquot The final volume is equal to the aliquot

volume PLUS the diluent volume: volume PLUS the diluent volume: 0.1 mL + 9.9 mL = 10 mL0.1 mL + 9.9 mL = 10 mL

The dilution factor is equal to the final The dilution factor is equal to the final volume divided by the aliquot volume: volume divided by the aliquot volume: 10 mL/0.1 mL = 100X dilution factor10 mL/0.1 mL = 100X dilution factor

Practice ProblemPractice Problem

What is the dilution factor when What is the dilution factor when 0.2 mL is added to 3.8 mL of 0.2 mL is added to 3.8 mL of diluent? diluent?

Serial DilutionsSerial Dilutions

If a 1/8 dilution of the stock If a 1/8 dilution of the stock solution is made followed by a 1/6 solution is made followed by a 1/6 dilution what is the final dilution?dilution what is the final dilution?

The final dilution is: 1/8 x 1/6 = The final dilution is: 1/8 x 1/6 = 1/481/48

DilutionsDilutions

Means to reduce a concentrationMeans to reduce a concentration Dilution: A fraction of the dilution Dilution: A fraction of the dilution

factorfactor

Ex. You have a solution at 25 mg/ml and wish to obtain a solution of 5mg/ml

Dilution factor = Conc. I have Conc. I want

Dilution factor = 25mg/mL 5mg/mL

= 5X

Dilution = 1/the dilution factor = 1/5 = 1 part/5 parts Total

ExampleExample

How would you prepare 25mL of a How would you prepare 25mL of a 2mM solution from a 0.1M stock?2mM solution from a 0.1M stock?

QuantitiesQuantities

Quantities are equal to amounts Quantities are equal to amounts NOT concentrations!NOT concentrations! Ex 1. Ex 1.

Two apples per bag = a concentrationTwo apples per bag = a concentration Two apples = an amountTwo apples = an amount

Ex 2.Ex 2. 10g per 100 mL = a concentration10g per 100 mL = a concentration 10g = an amount10g = an amount

From concentrations to From concentrations to amountsamounts

The concentration indicates the The concentration indicates the amount in a given volumeamount in a given volume Ex. 1mM = 1millimole per each literEx. 1mM = 1millimole per each liter Therefore the amount in 1 L is 1 Therefore the amount in 1 L is 1

millimolemillimole What volume of solution would you What volume of solution would you

need to have 0.05 millimoles?need to have 0.05 millimoles?

Agarose Gel Agarose Gel ElectrophoresisElectrophoresis

Separates single stranded or Separates single stranded or double stranded nucleic acid double stranded nucleic acid molecules according to their size molecules according to their size and their conformationand their conformation Separates fragments between 100pb Separates fragments between 100pb

and 10 Kbpand 10 Kbp Resolving power between fragments Resolving power between fragments

≥100pb≥100pb

Migration on an Agarose Migration on an Agarose GelGel

Well

Direction of migration

Top (-)

Bottom (+)

Supercoiled

Relaxed

Linear

What can be determined from What can be determined from an electrophoresis on an an electrophoresis on an agarose gel?agarose gel?

Is there DNAIs there DNA How many conformationsHow many conformations How many fragmentsHow many fragments

The size of the fragmentsThe size of the fragments Total size of nucleic acid moleculeTotal size of nucleic acid molecule The number of cutsThe number of cuts

Linear?Linear? Circular?Circular?

Migration Profile on Migration Profile on AgaroseAgarose

Migration distance

Log

of

the

size

1.0%

1.5%

Resolution

Resolution

Size (bp) Distance (mm)

23,000 11.0 9,400 13.0

6,500 15.0

4,400 18.0

2,300 23.0

2,000 24.0

Fingerprinting Standard Curve: Semi-logDetermining sizesDetermining sizes

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Molecular Biology Technical Skills. Skills Micropipetting Preparing solutions Working with concentrations Dilutions Amounts Agarose gel electrophoresis