Molecular Biology

Molecular Biology

Technical Skills

Skills

Micropipetting Preparing solutions Working with concentrations Dilutions Amounts Agarose gel electrophoresis

Micropipetting-Measuring small volumes

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

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

Setting 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 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 reproducibility

Use pipettor whose volume is closest to the one desired

Consistent SPEED and SMOOTHNESS to press and release the PLUNGER

Consistent IMMERSION DEPTH 3-4mm below surface

AVOID air bubbles NEVER go beyond the limits of the pipettor

Preparing Solutions

Definitions

Solution Mixture of 2 or more substances in a single

phase Solutions are composed of two constituents

SolutePart that is being dissolved or diluted – Usually

smaller amount Solvent (OR Diluent)

Part of solution in which solute is dissolved – Usually greater volume

Concentrations

Concentration = Quantity of solute Quantity of solution (Not

solvent)

Common ways to express concentrations: Molar concentration (Molarity) Percentages Mass per volume Ratios

Molarity

# of Moles of solute/Liter of solution

Mass of solute: given in grams (g) Molecular weight (MW): give in grams per

mole (g/mole)

Percentages

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

All represented as fractions of 100

Percentages (Cont’d)

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

Must have same units top and bottom!

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

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

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

Must have same units top and bottom!

Mass per volume

A mass (amount) per a volume Ex. 1kg/L Know the difference between an amount

and a concentration! In the above example 1 litre contains 1kg (an

amount)What amount would be contained in 100ml? What is the percentage of this solution?

Ratios

A way to express the relationship between different constituents

Expressed according to the number of parts of each component Ex. 24 ml of chloroform + 25 ml of phenol +

1 ml isoamyl alcohol Therefore 24 parts + 25 parts + 1 part Ratio: 24:25:1 How many parts are there in this solution?

Preparing solutions

Dilutions: Reducing a ConcentrationA Fraction

Dilutions

Dilution = making weaker solutions from stronger ones

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

Dilutions (cont’d)

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

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


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

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


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

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


Notice that dilutions do NOT have units (cans, ml, or parts) but are expressed as one number to another number Example: 1/10 or “one in ten” OR: 1/(1+9)


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


Example:Two (2) parts of dye are diluted with eight (8) parts of diluent (the term used for the diluting solution). The total solution volume is 10 parts (2 parts dye + 8 parts diluent). The dilution is initially expressed as 2/10, but the original substance must be expressed as one (1). To get the original volume to one (1), use a ratio and proportion equation, remembering that dilutions are stated in terms of 1 to something:______2 parts dye = ___1.0___ 10 parts total volume x

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

Problem

Two parts of blood are diluted with five Two parts of blood are diluted with five parts of salineparts of saline What is the dilution? What is the dilution?

10 ml of saline are added to 0.05 L of 10 ml of saline are added to 0.05 L of waterwater What is the dilution? What is the dilution?

2/(2+5) = 2/7 =1/3.5

10/(10+50) = 10/60=1/6

What Does This Mean??

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

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

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

Problem : More than one ingredient

Want to prepare 15 mL of a solution containing two ingredients (solutes) Need the following dilutions

Solute 1: 1/10 Solute 2: 1/3

Express each component being diluted over the same common denominator!

Solute 1: 1/10 = 1.5/15 Solute 2: 1/3 = 5/15 Therefore need 1.5 parts of solute 1 + 5 parts of

solute 2 + 8.5 parts solvant

Determining the Required Fraction:The Dilution

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

The fraction is equal to 1/the dilution factor = 1/5 (the dilution)

What I haveWhat I want

Determine the reduction factor (The dilution factor) =

Therefore the reduction factor is: 25mg/ml5mg/ml = 5 (Dilution factor)

Example

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

Determining the Volumes Required

Ex. You want 55 mL of a solution which represents a dilution of 1/5 Use a ratio equation: 1/5 = x/55 = 11/55

Therefore 11 mL of solute / (55 mL – 11 mL) of solvent

= 11 ml of solute / 44 ml of solvent

Problem #1

Prepare 25mL of a 2mM solution from a stock of 0.1M What is the dilution factor required? What is the dilution required? What volumes of solvent and solute are

required?

Solution #1

Fractions : 2mM = 0.002M (what I want) Stock = 0.1M (what I have)

Dilution factor = (what I want)/(what I have)Dilution factor = 0.1/0.002 = 50x

Required dilution = 1/Df = 1/50 Volume of a part = (Final volume)/(# of parts)

Volume of one part = 25mL/50 parts = 0.5mL/part

Solution #1 (Cont’d)

Volume of solute 1 part * 0.5mL/part = 0.5mL

Volume of solvent (50 – 1) parts * 0.5mL/part = 24.5mL

Quantities

Quantities are NOT concentrations! Ex 1.

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

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

From concentrations to amounts

The concentration indicates the amount in a given volume Ex. 1mM = 1 millimole per each liter Therefore the amount in 1 L is 1 millimole What volume of solution would you need to

have 0.05 millimoles?

Ratios

Means of expressing solutions by indicating the ration between the different components: Mass ratios Molar ratios Volume ratios

Mass ratios

Ex. 12g of NaCl is dissolved in 1000ml of water Convert the units so that they are the same

12g of NaCl in 100g of water Divide the quantities by the value of the

smallest quantity 12g/12g : 100g/12g

The ratio NaCl : water= 1:8.3

Molar ratiosEx. 12g of NaCl is dissolved in

100ml water Convert the units into moles

12g/(58g/mole) of NaCl in 100g/(18g/mole) of water

– 0.2 moles of NaCl : 5.6 moles of water

Divide the quantities by the value of the smallest quantity

0.2moles/0.2moles: 5.6moles/0.2moles The ratio

NaCl : water= 1:28

Volume ratiosVolume ratios

Ex. 12ml of alcohol are added to 1L Ex. 12ml of alcohol are added to 1L of waterof water Convert the units so that they are the Convert the units so that they are the

samesame 12ml alcohol in 1000ml of water12ml alcohol in 1000ml of water

Divide the qauntities by the value of Divide the qauntities by the value of the smallest quantitythe smallest quantity

12ml/12ml : 1000ml/12ml12ml/12ml : 1000ml/12ml The ratioThe ratio

Alcohol : water = 1:83.3Alcohol : water = 1:83.3

Agarose gel electrophoresis

Separation of single or double stranded nucleic acids according to their size and conformation Separation of fragments between 100pb

and 10 Kbp Resolution of fragments ≥100pb

Undigested plasmid on a gel

Undigested plasmids generate a pattern of bands

Migration is a function of size and conformation Supercoiled Relaxed Multimers?

Supercoiled

-

+

Relaxed

multimers

-

Migration of linear DNA-Digested plasmids

The migration speed is a function of the size

Smaller fragments migrate fasterThe migration speed is inversely

proportional to the log10 of the size

Migration of linear DNA-digested plasmids

1000 bp850 bp750 bp600 bp

200 bp100 bp

-

Sam

ple 1

Sam

ple 2

+

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 sizes

Visualization: Ethidium Bromide

Stain used to make nucleic acids visible Fluorescent under UV Binding is proportional to

The size The quantity The conformation

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

Is there DNA How many conformations How many fragments

The size of the fragments Total size of nucleic acid molecules The number of cuts

Linear? Circular?

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Molecular Biology