Lectures 1-5

Question 1

Scientific method:

Answer 1

Observation - hypothesis - experiment - conclusion - scientific theory

Question 2

Variables:

Answer 2

Controlled, independent, dependent

Question 3

Experimental controls:

Answer 3

Positive - a treatment that gives the desired result

Negative - a treatment that does not give the desired result

Question 4

False results:

Answer 4

Positive - from negative control; desired result when it shouldn’t be
Negative - from positive control; lack of desired result

Question 5

Concerns to consider:

Answer 5

Experimenter/subject biases, record of procedure, reproducibility, qualitative vs quantitative data, statistical significance, correlation vs causation

Question 6

Blind studies - single vs double:

Answer 6

Single blind - experimenter doesn’t know which treatment the subject is under; rules out coaching.
Double blind - neither experimenter nor subject knows; rules out coaching and placebo.

Question 7

Define: sensitivity

Answer 7

Minimum amount of X needed to record a positive result. Ex: weighing sand on a bathroom scale.

Question 8

Define: specificity

Answer 8

A positive result only comes from a truly positive sample. Ex: Measuring UV rays - instrument that measures all light vs instrument that measures UVa, UVb.

Question 9

Define: random error

Answer 9

New error introduced with each measurement. Based on lab practices.

Question 10

Define: systematic error

Answer 10

Error that is consistently present. Remember to calibrate!

Question 11

Define: accuracy

Answer 11

How close a recorded value is to the true value

Question 12

Define: precision

Answer 12

How reliably you can measure a value

Question 13

pH meter:

Answer 13

Specifically permeable to hydrogen. Current produced by H+ is compared to standards of pH value. Stored in KCl or acidic buffer.

Question 14

Random/systematic error in using pH meters:

Answer 14

Random: not cleaning probe properly (carry over contaminants), everything is mixed properly, reading the instrument properly (letting the instrument settle on a number)

Systematic error: not calibrating properly

Question 15

Sensitivity and random error:

Answer 15

More sensitivity means more random error.

Question 16

Accuracy/precision vs random/systematic:

Answer 16

Accuracy is more affected by systematic error, precision is more affected by random error.

Question 17

Vortex mixer:

Answer 17

Used to mix solutions in Eppendorf tubes (1.5 mL). Different settings for different needs - some solutions need a lot of force, while others will shear apart.

Question 18

Stir plate:

Answer 18

Teflon coated magnets placed in solution; magnet in base with adjustable spin speed.
Sometimes coupled with a heating element to aid in dissolving solutes - don’t kill your sample tho

Question 19

Micropipettes and their random/systematic errors:

Answer 19

10-0.0002 mL.
Random - air bubbles
Systematic - using outside range

Question 20

Standard molarity of water:

Answer 20

55.5 mol/L

Question 21

What is pKa?

Answer 21

When there are equal amounts of acid and conjugate base.

Question 22

Henderson-Hasselbalch equation:

Answer 22

pH = pKa + log [A-]/[HA]

DO PRACTICE PROBLEMtS.

Question 23

Double-checking the H-H:

Answer 23

If pH

Question 24

What happens at +/- 1 pH from pKa? What doesn’t happen?

Answer 24

Buffer stops being efficient. It’s not depleted, it’s just really bad at being a buffer.

Question 25

Charges at pKa:

Answer 25

HALF CHARGES

Question 26

Variables for effectiveness of heat killing:

Answer 26

Temperature, time, conductance

Question 27

Autoclave:

Answer 27

High pressure increases boiling point. High boiling point increases temperature of steam.
15 PSI = 121ºC

Question 28

Filtration:

Answer 28

Suction through filter with

Question 29

Energy and wavelength:

Answer 29

High energy = short wavelength (violet)

Low energy = long wavelength (red)

Question 30

Transmittance and absorbance equation:

Answer 30

A = log (1/T)

Question 31

Choosing optimal wavelength from absorbance:

Answer 31

Peaks that don’t overlap with other ones.

Question 32

Beer-Lambert equation:

Answer 32

A = eLc

e has inverse units of L and c

Question 33

[DNA] from absorbance:

Answer 33

Abs * dilution factor * 50 = [DNA]

50 is the e estimate for DNA.

Question 34

Estimate of e for RNA?

Answer 34

Not 50, like DNA. Something like 40.

Question 35

DNA and RNA are hard to measure directly in a spec because:

Answer 35

They are quite transparent. Absorb UV at 260 nm. Cell wall redirects light, which counts as being absorbed.

Question 36

Measuring bacteria in a spec:

Answer 36

595-600 nm.

Question 37

Measuring proteins in a spec:

Answer 37

Absorb UV at 280 nm. AA sequence matters for absorbance - it’s quite sensitive.

Question 38

Determining DNA purity:

Answer 38

Compare peaks of DNA vs protein. A260/A280 of pure DNA is 2 (but 1.8 is okay). A260/A280 of pure protein is 0.55.
The beginning of the curve is hard to get reproducible measurements from, so 1/3 DNA and 2/3 protein is good.

Question 39

Bradford assay:

Answer 39

Indirect protein assay. Measurement of colour change when Coomassie Blue binds protein. Unbound dye has a max abs of 465 nm; bound dye has a max abs of 595 nm.
Thumbs down: Reaction varies with AA sequence (likes basic ones) and protein function. Upper limit for detection is 1 mg/mL. Dye may precipitate with detergent.

Question 40

Biuret assay:

Answer 40

Indirect protein assay. Measurement of colour change when CuSO4 reacts with peptide bonds under alkaline solutions. Turns purple (540 nm). Useful to 10 mg/mL.
Thumbs down: takes 15 minutes.

Question 41

Compare Bradford and Biuret:

Answer 41

Biuret is better because it has a larger range. Biuret is more specific - Bradford would tell you there are proteins in a solution when there are actually just AAs. Biuret takes longer.