General Information Instructor: John Basso Email: jbasso@uottawa.ca Tel. 613-562-5800 Ext. 6358 Office: BSC102 My web page : http://mysite.science.uottawa.ca/jbasso/home.htm Course’s web page: http://mysite.science.uottawa.ca/jbasso/molecular/home.htm

My Web Page

Grading Quiz – 5% Each week Available on Brightspace Saturday 9 am to Sunday 9 am 30 minutes 2% bonus for 100% on at least 4 of the 8 quizzes Quiz 1 Saturday January 13th

Grading Assignments X 5 - 25% Submitted individually or in groups of two Part I: Theory Part II: Data analysis Lab performance Submit your own results Part III: Bioinformatics

Grading Exams Open book Access to the internet is allowed for midterm Use of laptops is not allowed Must use lab computers

Grading Midterm – 10-25% 2 hours 5 calculation problems (5 points) 5 bioinfo exercises (5 points) 5 theoretical questions on bioinfo and molecular procedures (5 points) 2 out of 3 problems on data analysis and experimental design (10 points)

Grading Final exam – 45-60% Cumulative 4 hours Part I – 1.5 hours 10 bioinfo exercises (10 points) Part II – 2.5 hours 10 calculation problems (10 points) 5 theoretical questions on bioinfo and molecular procedures (5 points) 3 out of 4 problems with an emphasis on data analysis and experimental design (15 points)

Grading Quiz 5% + *Bonus 2% Assignments (X5) 25% Midterm exam 10%   Option I Option II Quiz 5% + *Bonus 2% Assignments (X5) 25% Midterm exam 10% Final exam 45% 60% Total 102%

Obtaining your data You must have a USB key to save your pictures Absorbance readings will be available on the web It is recommended that you have a note book

Working with Solutions

Definitions Solution Mixture of 2 or more ingredients in a single phase Solutions are composed of two constituents Solute (ingredient) A solid that is being dissolved Or a stock solution that is being diluted Solvent (OR Diluent) Part of solution in which solute is dissolved or stock solution is being diluted

Preparing solutions 2 ways to create a solution By dissolving a solid By diluting a more concentrated solution Do your calculations Final volume required Mass of solid (solutes) Volume of stock solutions Volume of solvant  Add solvent first (usually water) Add other ingredients to solvent If enzymes are required add last

Measuring small volumes - Micropipetting Allows to measure microliters (µL) 1 000 X less than 1 milliliter 2-20 µL 50-200 µL 100-1000 µL Max. 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)

Setting the volume- P200 Hundreds (0, 1=100 or 2=200) Tens (0, 1-9=10-90) Units (1-9)

Setting the volume- P1000 Thousands (0, 1=1000) Hundreds (0, 1-9=100-900) Tens (0, 1-9=10 - 90)

Using the micropipettor Step 1 Insert tip Step 3 Insert tip in solution to be drawn Step 4 Draw up sample by slowly releasing plunger Step 2 Press plunger up to first stop Step 5 Withdraw 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 Working with concentrations Dilutions Amounts

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): given in grams per mole (g/mole) Moles of solute = Mass of solute MW of solute Molarity = Moles of solute Volume in L of solution

Percentages Percentage concentrations can be expressed as either: V/V – volume (mL) of solute/100 mL of solution M/M – Mass of solute (g) /100 g of solution M/V – Mass of solute (g) /100 mL of solution All represented as fractions of 100

Percentages (Cont’d) %V/V %M/V % M/M 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%

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?

Dilutions: Reducing a Concentration A Fraction Preparing solutions Dilutions: Reducing a Concentration A 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”.

Dilutions (cont’d) 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).

Dilutions (cont’d) 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”.

Dilutions (cont’d) Notice that dilutions do NOT have units (cans, ml, or parts) but are expressed as the number parts to the total number of parts Dilutions are always expressed as a fraction of one 1 part / total number of parts Example: 1/10 or “one in ten” OR: 1/(1+9) OR 1 part solute/1 part solute + 9 parts solvent

Dilutions (cont’d) Dilutions are always expressed with the original substance being 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.

Dilutions (cont’d) Example: Two (2) parts of dye are diluted with eight (8) parts of solvant. 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 = 5 The dilution is expressed as 1/5.

Problem Two parts of blood are diluted with five parts of saline What is the dilution? 10 ml of saline are added to 0.05 L of water 2/(2+5) = 2/7 =1/3.5 10/(10+50) = 10/60=1/6

Working with parts Preparation of 110 mL solution representing a 1/10 dilution 1/10th of final volume must be stock solution 9/10th of final volume must be solvent 1/10th of 110 mL = 11 mL = 1 part 9/10th of 110 mL = 99 mL = 9 parts

Working with parts A solution is prepared by adding 15 mL of a stock solution to 75 mL of solvent. What is the dilution and the volume of 1 part? Volume of one part Fraction: 15mL stock/(15mL stock + 75mL solvent) = 15/90 = 1/6 dilution Solution is therefore composed of a total of 6 parts Total number of parts = 6 = 90 mL One part = 90 mL/6 = 15 mL

Problem : More than one ingredient Want to prepare 25 mL of a solution containing two ingredients (solutes) Need the following dilutions Solute “a”: 1/10 Solute “b”: 1/3

Problem: More than one ingredient Express each ingredient being diluted over the same common denominator! Solute « a » : 1/10 = 1.5/15 Solute « b » : 1/3 = 5/15 Therefore need 1.5 parts of solute “a” + 5 parts of solute “b” + 8.5 parts of solvent Thus a total of 6.5 parts solute/15 parts solution 15 parts of solution = 25mL, thus 1 part = 1.67 mL Need 1.5 parts of solute “a” :1.5 X 1.67 mL = 2.51 mL Need 5 parts of solute “b”: 5 X 1.67 mL = 8.35 mL Need 8.5 parts of solvent: 8.5 X 1.67 mL = 14.20 mL

Determining the Required Fraction: The Dilution What I have What I want Determine the reduction factor (The dilution factor) = Ex. You have a solution at 25 mg/ml and want to obtain a solution at 5mg/ml Therefore the reduction factor is: 25mg/ml 5mg/ml = 5 (Dilution factor) The fraction is equal to 1/the dilution factor = 1/5 (the dilution)

Determining the Volumes Required Ex. You want 55 mL of a solution which represents a 1/5 dilution Use a ratio equation: 1/5 = x/55 = 11/55 Therefore 11 mL of stock/ (55 mL – 11 mL) of solvent = 11 ml of stock/ 44 ml of solvent

Problem #1 Prepare 25mL of a 2mM solution from a stock of 0.1M What is the dilution required? What volumes of solvent and stock solution are required? 2mM/100mM =1/50

Problem #2 What volume of a 0.1M stock solution should be added to 25 mL of water to obtain a 2mM final concentration ? What is the dilution required? Volumes of solute required? 2mM/100mM = 1/50 1/50 dilution = 1 part stock/50 parts solution = 1 part stock/1 part stock + 49 parts solvent Volume of solvent = 25 mL = 49 parts Therefore volume of one part = 25 mL/49 = 0.51mL 0.51/(25 +0.51) = 0.51/25.51 = 1/50

Quantities Quantities are NOT concentrations! Ex 1. Ex 2. 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 ratio 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 ratios Ex. 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 ratios Ex. 12ml of alcohol are added to 1L of water Convert the units so that they are the same 12ml alcohol in 1000ml of water Divide the qauntities by the value of the smallest quantity 12ml/12ml : 1000ml/12ml The ratio Alcohol : water = 1:83.3

Densities Densities are not concentrations! Density expresses the relationship between a mass and the volume it occupies g/mL Example: 1g of water occupies a volume of 1 mL Note: The density of a solute (solid) is not the same as that of a dissolved solute (solution)

Densities Example Sample problem: Density of NaCl: 2.16 g/mL Denisty of 10% NaCl (m/v): 1.07 g/mL Sample problem: How much water is there in 100 mL of a 30% (m/v) NaCl solution 30g NaCl /100 ml solution 30g NaCl occupies a volume of 13.89 mL Therefore volume of water = 100 – 13.89 = 86.11 mL