Molecular Biology Technical Skills
Skills • Micropipetting • Preparing solutions • Working with concentrations • Dilutions • Amounts • Agarose gel electrophoresis
2-20 µL 50-200 µL 100-1000 µL Max. 0.02 mL 0.2mL 1mL Micropipetting- Measuring small volumes • Allows to measure microliters (µL) • 1 000 X less than 1 milliliter
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)
Step 3 Insert tip in solution to be drawn Step 1 Insert tip Step 2 Press plunger up to first stop Step 5 Withdraw pipettor Using the micropipettor Step 4 Draw up sample by slowly releasing plunger
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
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
Concentration = Quantity of solute Quantity of solution (Not solvent) Three basic ways to express concentrations: Molar concentration (Molarity) Percentages Mass per volume Ratios Concentrations
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
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 represent 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!
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 one number to another number • Example: 1/10 or “one in ten”
Dilutions (cont’d) • 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.
Dilutions (cont’d) 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 = 5 The dilution is expressed as 1/5.
Dilutions (cont’d) The dilution does not always end up in whole numbers. Example: Two parts (2) parts of whole blood are diluted with five (5) parts of saline. The total solution volume is seven (7) parts (2 parts of whole blood + 5 parts saline). The dilution would be 2/7, or, more correctly, 1/3.5. Again, this is calculated by using the ratio and proportion equation, remembering that dilutions are stated in terms of 1 to something: __2 parts blood_____ = ___1.0___ 7 parts total volume x 2 x = 7 x = 3.5 The dilution is expressed as 1/3.5
What Does This Mean?? • If a solution has 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?
Determining the dilution required • Dilution: A fraction of the dilution factor Dilution factor = Conc. I have Conc. I want Ex. You have a solution at 25 mg/ml and wish to obtain a solution of 5mg/ml Dilution factor = 25mg/mL 5mg/mL = 5X Dilution = 1/the dilution factor = 1/5 = 1 part/5 parts Total
Example • How would you prepare 25mL of a 2mM solution from a 0.1M stock?
Quantities • Quantities are NOT concentrations! • Ex 1. • Twoapples per bag = a concentration • Twoapples = 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 = 1millimole per each liter • Therefore the amount in 1 L is 1 millimole • What volume of solution wouldyouneed 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 NaClisdissolved in 1000ml of water • Convert the unitssothatthey are the same • 12g of NaCl in 100g of water • Divide the quantities by the value of the smallestquantity • 12g/12g : 100g/12g • The ratio • NaCl : water= 1:8,3
Molar ratios • Ex. 12g of NaClisdissolved in 100ml water • Convert the unitsinto 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 smallestquantity • 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 unitssothatthey are the same • 12ml alcohol in 1000ml of water • Divide the qauntities by the value of the smallestquantity • 12ml/12ml : 1000ml/12ml • The ratio • Alcohol : water = 1:83.3
Agarose gel electrophoresis • Separation of single or double strandednucleicacidsaccording to their size and conformation • Separation of fragments between 100pb and 10 Kbp • Resolution of fragments ≥100pb
Supercoiled Relaxed - Undigested plasmid on a gel - • Undigestedplasmidsgenerate a pattern of bands • Migration is a function of size and conformation • Supercoiled • Relaxed • Multimers? multimers +
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
1000 bp 850 bp 750 bp 600 bp 200 bp 100 bp Migration of linear DNA (digested plasmids) Sample 1 Sample 2 - +
Determining sizes Fingerprinting Standard Curve: Semi-log 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
Visualization: Éthidium bromide • Stainused to makenucleicacids visible • Fluorescent under UV • Bindingisproportional 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?