1. Today Housekeeping Lab Math Preparing reagents Field trip Topics
Website (
Homework
Lab Math
Preparing reagents

2. Field trip Sevilleta

3. TOPICS/WEBSITE

4. 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

5. 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

7. 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,

8. 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.

9. 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

10. 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)

11. 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

12. 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

13. 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

14. 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

15. 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”

16. 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

17. 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)

18. 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

19. 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

20. 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

21. 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.

22. Example
How are 50 ml of 20 mM NaOH made?

23. 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

24. 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%)

25. 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)