ppt课件-lagénomique-universityofottawa.pptVIP

Quantities Quantities are equal to amounts 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 = 1millimole per each liter Therefore the amount in 1 L is 1 millimole What volume of solution would you need to have 0.05 millimoles? Agarose Gel Electrophoresis Separates single stranded or double stranded nucleic acid molecules according to their size and their conformation Separates fragments between 100pb and 10 Kbp Resolving power between fragments ≥100pb Migration on an Agarose Gel Well Direction of migration Top (-) Bottom (+) Supercoiled Relaxed Linear 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 molecule The number of cuts Linear? Circular? Migration Profile on Agarose Migration distance Log of the size 1.0% 1.5% Resolution Resolution 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-log Determining sizes * * * * * * * * * 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 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 2 Press plunger up to first stop Step 3 Insert tip in solution to be drawn Step 4 Draw up sample by sl