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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)
Setting the volume- P200Setting the volume- P200
Hundreds (0, 1=100 or 2=200)
Tens (0, 1-9=10-90)
Units (1-9)
Setting the volume- P1000Setting the volume- P1000
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
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
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
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”.
Dilutions (cont’d)Dilutions (cont’d)
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).
Dilutions (cont’d)Dilutions (cont’d)
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”.
Dilutions (cont’d)Dilutions (cont’d)
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 (cont’d)Dilutions (cont’d)
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.
Dilutions (cont’d)Dilutions (cont’d)
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.
Dilutions (cont’d)Dilutions (cont’d)
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
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