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Today Housekeeping Lab Math Preparing reagents Field trip Topics
Website ( Homework Lab Math Preparing reagents
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Field trip Sevilleta
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TOPICS/WEBSITE
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PARASITES AND SNAIL BIOLOGY
DNA “identity, possibilities” phylogenetics RNA “intentions” transcriptomics CTAB/DNAZOL Trizol gel electrophoresis nanodrop spec Bioanalyzer DNA-free, PCR rDNA/mito TA cloning, B/W screening RT-PCR gel electrophoresis Qiagen plasmid extraction Restriction digests direct sequencing M13 sequencing Sequence ID (BLAST) editing Primer design, walking Phylogenetics GenBank submission 4
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Preparing reagents for upcoming experiments
CTAB solution for DNA extraction 76% ethanol/10 mM ammonium acetate 0.25 M EDTA 3 M Na acetate (CH3COONa or NaOAc) 50X TAE Electrophoresis sample buffer
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Standard versus Metric or the International System of Units,
DIFFERENT UNITS, STANDARD OR SI NL mile or km 1 kilometer is mile; 1 mile is 1.6 kilometer; 100 kilometer/h is 62.5 mph Standard versus Metric or the International System of Units,
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International System of Units.
The SI (from Le Système International d'Unités), is the modern metric system of measurement, founded on 7 mutually independent base units HISTORY Decimal Metric System (1799, French Revolution) platinum standards representing the meter and the kilogram. 1832: millimeter, gram and second for length, mass and time 1889: base units changed to meter, kilogram and second 1954 addition of ampere, kelvin and candela for electric current, thermodynamic temperature and luminous intensity 1960: The system was named International System of Units (SI) by the international General Conference on Weights and Measures (CGPM, Conférence Générale des Poids et Mesures). The SI is a universal language of science, adopted by trade (not everywhere). It is modified to reflect the latest advances in science and technology. 1971 the 7th base unit completed the current SI by addition of the mole as base unit for amount of substance.
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Mole and molecular weight (MW)
1 MOLE = The amount of a substance that contains as many atoms, molecules, ions, or other elementary units as the number of atoms in kilogram of carbon 12. The number is × 1023, or Avogadro's number. The mass in grams of this amount of a substance is numerically equal to the molecular weight of the substance. Also called gram-molecular weight (MW). 1 Carbon (12C) atom: 12g/ × 1023 atoms = × g or g MW can be calculated from chemical formulas using atomic mass It is given in reference books or on jars So we can quantify (measure) amounts of atoms, chemicals 1Mole of 12C = 12g, 0.5 Mole of 12C = 6g
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One mole of marshmallows (6
One mole of marshmallows ( × 10^23) would have a mass of 17 exatonnes (1.7 x 10^25 g or 3.75 x 10^22 lbs), and cover up Earth approximately 100 km (60miles) deep (wikipedia)
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Prefixes most used in molecular biology
Abbreviation Power of 10 giga G 109 mega M 106 kilo k 103 - 100 milli m 10-3 micro 10-6 nano n 10-9 pico p 10-12 femto f 10-15 atto a 10-18
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Metric calculations with moles or the stair of 1000s
millimole 1000 micromole 1000 nanomole 1000 picomole 1 mole = 1,000 mmole = 1,000,000 μmole 1 mole = 103 mmole = 106 μmole 1 pmole = nmole/1000 = μmole/1,000,000 1 pmole = 10-3 nmole = 10-6 μmole
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MOLARITY MOLAR = CONCENTRATION the number of moles of solute/ liters of solution (ALWAYS consider LITERS) 1 molar = 1 M = 1 mole/L To use a chemical at a particular concentration, take any volume (the concentration does not change!) To use an amount of a chemical solubilized (dissolved) at a particular concentration, take part of a liter: 1mole/L, 1mmole/mL, 1mmole/mL Always ask do I need an amount or a concentration? amount relates to weight Concentration relates to weight/LITER
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Working solutions Usually made from concentrated stock solutions
Minimize work, modest storage space Dilute stock to working solution STOCKS and lab etiquette MUST BE MADE CORRECTLY TEND TO RUN OUT WHEN NEEDED
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Calculating Dilutions
Need 125 mM EDTA Have stock of 0.5 M EDTA (pH 8.0) What do you do? “SEE IT” or “CALCULATE IT”
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C1V1=C2V2 Convert everything to same units
Plug in the numbers (sometimes confusing) If the answer doesn’t seem right (amount to dilute is > than final volume), your setup wasn’t right and redo In general, if something doesn’t seem right, then it probably isn’t
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Calculating Dilutions
Need 125 mM EDTA Have 0.5 M EDTA Solution: C1V1=C2V2 500 mM X 1 ml = 125 mM X z ml 500 mM X 1 ml = 125 mM X z ml 125 mM mM 4 x 1 ml = z ml, z =4 Take 1 ml 500 mM, add 3 ml water (dilute 4 x)
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Concentrations by a factor of X
Expressed as a multiple of its standard working concentration Many solutions are 10 or 50 fold more concentrated than working solution Need to make it into 1X solution Say you wanted to make 1 L of 1X buffer from 10X stock: 10X buffer * n ml = 1X buffer *1000 ml 10*n =1000, n= 1000/10, n=100ml Take 100ml 10X and increase to 1L
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Oddities 1% (v/v) 1 ml/100ml 1% (w/v) 1 gr/100ml
1N (Normal): number of hydrogen ions per mole of a substance that contribute to pH 1N = 1M HCL, = 0.5M H2SO4, = 1M NaOH Dilutions: 1/10 = 1+9 because 1+10 = 1/11
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Preparing percent solutions
Prepared as a percent solute (e.g. salt) MW not considered Percent means per 100 g/100 ml 10% (w/v) means 10g/100 ml solution
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Diluting percent solutions
Example: Make 30 ml of 70% ethanol from 95% stock, how much of the stock into 30 ml? 0.95 X a ml = 0.70 X 30 ml a ml = 0.70 x 30 ml/0.95 = 19.95ml Take ml of 95% stock, add to 30 with water.
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Example How are 50 ml of 20 mM NaOH made?
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Solution Convert to mM to M and ml to L What is the MW of NaOH
(hint: look on the bottle) 0.05 L X 0.02 M X 40g/mole 0.05 L X 0.02 mole/L X 40g/mole Check your answer Make sure that everything cancels out and you are left with the proper units 0.04g
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First 80 then 100 Dissolve all components in 80% of the final volume
Volume contributed by chemicals, pH adjustment. Once dissolved, adjusted, add up to final volume (100%)
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Preparing reagents for upcoming experiments
CTAB solution for DNA extraction 76% ethanol/10 mM ammonium acetate 0.25 M EDTA 3 M Na acetate (NaOAc) 50X TAE Electrophoresis sample buffer (HOMEWORK QUESTIONS)
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