1. General Information Instructor: John Basso
Tel Ext. 6358
Office: BSC102
My web page :
Course’s web page:

2. My Web Page

3. Grading Quiz – 5% Each week, starting on week 2
Available on Blackboard on the Saturday before the following week’s lab between 9 am and 9 pm
30 minutes
2% bonus for 100% on at least 4 of the 8 quizzes

4. Grading Assignments - 15% Submitted individually or in groups of two
4 parts
Part I: Theory
To be submitted on Blackboard
Part II: Assignments on procedures and the data obtained.
Part III: Bioinformatics
Part IV: Lab performance – 10%
Submit your own results

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

6. Grading Midterm – 15-25% 8 calculation problems (16 points)
6 bioinfo exercises (6 points)
5 theoretical questions on bioinfo and molecular procedures (5 points)
2 out of 3 problems on data analysis and experimental design (8 points)

7. Grading Final exam – 45-55% Cumulative
5 calculation problems (10 points)
10 bioinfo exercises (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)

8. Grading Quiz 5% + *Bonus 2% Assignments (X5) 15% Lab performance 10%
Option I
Option II
Quiz
5% + *Bonus 2%
Assignments (X5)
15%
Lab performance
10%
Midterm exam
25%
Final exam
45%
55%
Total
102%

9. 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

10. Molecular Biology
Preparing Solutions

11. Preparing solutions
Working with concentrations
Dilutions
Amounts

12. Definitions Solution Mixture of 2 or more substances in a single phase
Solutions are composed of two constituents
Solute
Part that is being dissolved or diluted – Usually smaller amount
Solvent (OR Diluent)
Part of solution in which solute is dissolved – Usually greater volume

13. Concentrations Concentration = Quantity of solute
Quantity of solution (Not solvent)
Common ways to express concentrations:
Molar concentration (Molarity)
Percentages
Mass per volume
Ratios

14. 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)

15. 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

16. 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%

17. 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?

18. 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?

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

20. 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.

21. 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”.

22. 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).

23. 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 parts water). The dilution is written as 1/101 or said “one in one hundred and one”.

24. Dilutions (cont’d)
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)
OR 1 part solute/1 part solute + 9 parts solvent

25. 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.

26. 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 =
x =
The dilution is expressed as 1/5.

27. 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

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

29. Working with parts
A solution is prepared by adding 15 mL of a solute to 75 mL of solvent. What is the dilution and the volume of 1 part?
Fraction: 15mL solute/(15mL solute + 75mL solvent)
Volume of 1 part: total volume 90 mL = 6 parts
= 15/90 = 1/6 dilution or 1 part/1 part + 5 parts
90/6 = 1 parts = 15ml

30. Problem : More than one ingredient
Want to prepare 15 mL of a solution containing two ingredients (solutes)
Need the following dilutions
Solute “a”: 1/10
Solute “b”: 1/3

31. 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 » parts of solvent
Thus a total of 6.5 parts solute/15 parts solution
15 parts of solution = 15mL, thus 1 part = 1 mL
Need 1.5 parts of solute “a” :1.5 X 1 mL = 1.5mL
Need 5 parts of solute “b”: 5 X 1 mL = 5 mL
Need 8.5 parts of solvent: 8.5 X 1 mL = 8.5 mL

32. 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: mg/ml
5mg/ml =
5 (Dilution factor)
The fraction is equal to 1/the dilution factor = 1/5 (the dilution)

33. 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

34. 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?
1/50

35. 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 factor required?
What is the dilution required?
Volumes of solute required?
Df = 100mM/2mm =50X

36. 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

37. 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?

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

39. 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

40. 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

41. 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

42. 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

43. Undigested plasmid on a gel-
Undigested plasmid on a gel -
Undigested plasmids generate a pattern of bands
Migration is a function of size and conformation
Supercoiled
Relaxed
Multimers?
multimers
Relaxed
Supercoiled +

44. Migration of linear DNA-Digested plasmids
The migration speed is a function of the size
Smaller fragments migrate faster
The migration speed is inversely proportional to the log10 of the size

45. Migration of linear DNA-digested plasmids
Sample 1
Sample 2 -
1000 bp
850 bp
750 bp
600 bp
200 bp
100 bp +

46. Determining sizes
Fingerprinting Standard Curve: Semi-log
Size (bp) Distance (mm)
23, 9, 6, 4, 2, 2,

47. Visualization: Ethidium Bromide
Stain used to make nucleic acids visible
Fluorescent under UV
Binding is proportional to
The size
The quantity
The conformation

48. 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?