Today• Housekeeping

Field trip

TopicsWebsite (http://biology.unm.edu/cmadema/4546/4546.htm)

Homework

• Lab Math• Preparing reagents

Field trip Sevilleta

• Discovery of DNA structure

• Restriction enzymes

• Southern blotting

• Cloning

• The first sequenced gene

• Sensitivity of PCR

• RAPDs

• q-PCR

• BAC libraries

• ESTs

• BLAST and database searches

• Microarrays

• Forensics

• Genome sequencing , the $1000 genome

• Next generation sequencing

• Bioinformatics

• Epigenetics

• "non-coding" RNA

• C-value paradox

• Phylogenetic genomics

• Archeological genomics

• YOUR favorite gene (check with instructor)

TOPICS/WEBSITE

PARASITES AND SNAILPARASITES AND SNAIL BIOLOGY BIOLOGY

“identity, possibilities”phylogenetics

“intentions”transcriptomics

PCRrDNA/mito

BioanalyzerDNA-free,

direct sequencing

gel electrophoresisnanodrop spec

Sequence ID (BLAST)editing

Phylogenetics

electrophoresisRT-PCR

gel

CTAB/DNAZOL

Trizol

TA cloning, B/W screening

M13 sequencing

Primer design, walking

Qiagen plasmid extraction Restriction digests

DNA

RNA

GenBank submission

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

DIFFERENT UNITS, STANDARD OR SI

1 kilometer is 0.625 mile; 1 mile is 1.6 kilometer; 100 kilometer/h is 62.5 mphStandard versus Metric or the International System of Units,

NL

mile or km

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.

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 0.012 kilogram of carbon 12. The number is 6.0225 × 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/ 6.0225 × 1023 atoms = 1.9926 × 10-23 g or 0.000000000000000000000019926 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, chemicals1Mole of 12C = 12g, 0.5 Mole of 12C = 6g

One mole of marshmallows (6.0225 × 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)

Prefix Abbreviation Power of 10

giga G 109

mega M 106

kilo k 103

- - 100

milli m 10-3

micro µ 10-6

nano n 10-9

pico p 10-12

femto f 10-15

atto a 10-18

Prefixes most used in molecular biology

Metric calculations with moles or the stair of 1000s

Mole

millimole

micromole

nanomole

picomole

1000

1000

1000

1000

1 mole = 1,000 mmole = 1,000,000 μmole1 mole = 103 mmole = 106 μmole1 pmole = nmole/1000 = μmole/1,000,0001 pmole = 10-3 nmole = 10-6 μmole

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, 1mole/L

• Always ask do I need an amount or a concentration?• amount relates to weight• Concentration relates to weight/LITER

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

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”

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

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 125 mM• 4 x 1 ml = z ml, z =4• Take 1 ml 500 mM, add 3 ml water (dilute 4 x)

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

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

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

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 19.95 ml of 95% stock, add to 30 with water.

Example

• How are 50 ml of 20 mM NaOH made?

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

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%)

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)