Lectures 1-5 Flashcards
Scientific method:
Observation - hypothesis - experiment - conclusion - scientific theory
Variables:
Controlled, independent, dependent
Experimental controls:
Positive - a treatment that gives the desired result
Negative - a treatment that does not give the desired result
False results:
Positive - from negative control; desired result when it shouldn’t be
Negative - from positive control; lack of desired result
Concerns to consider:
Experimenter/subject biases, record of procedure, reproducibility, qualitative vs quantitative data, statistical significance, correlation vs causation
Blind studies - single vs double:
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.
Define: sensitivity
Minimum amount of X needed to record a positive result. Ex: weighing sand on a bathroom scale.
Define: specificity
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.
Define: random error
New error introduced with each measurement. Based on lab practices.
Define: systematic error
Error that is consistently present. Remember to calibrate!
Define: accuracy
How close a recorded value is to the true value
Define: precision
How reliably you can measure a value
pH meter:
Specifically permeable to hydrogen. Current produced by H+ is compared to standards of pH value. Stored in KCl or acidic buffer.
Random/systematic error in using pH meters:
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
Sensitivity and random error:
More sensitivity means more random error.
Accuracy/precision vs random/systematic:
Accuracy is more affected by systematic error, precision is more affected by random error.
Vortex mixer:
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.
Stir plate:
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
Micropipettes and their random/systematic errors:
10-0.0002 mL.
Random - air bubbles
Systematic - using outside range
Standard molarity of water:
55.5 mol/L
What is pKa?
When there are equal amounts of acid and conjugate base.
Henderson-Hasselbalch equation:
pH = pKa + log [A-]/[HA]
DO PRACTICE PROBLEMtS.
Double-checking the H-H:
If pH
What happens at +/- 1 pH from pKa? What doesn’t happen?
Buffer stops being efficient. It’s not depleted, it’s just really bad at being a buffer.
Charges at pKa:
HALF CHARGES
Variables for effectiveness of heat killing:
Temperature, time, conductance
Autoclave:
High pressure increases boiling point. High boiling point increases temperature of steam.
15 PSI = 121ºC
Filtration:
Suction through filter with
Energy and wavelength:
High energy = short wavelength (violet)
Low energy = long wavelength (red)
Transmittance and absorbance equation:
A = log (1/T)
Choosing optimal wavelength from absorbance:
Peaks that don’t overlap with other ones.
Beer-Lambert equation:
A = eLc
e has inverse units of L and c
[DNA] from absorbance:
Abs * dilution factor * 50 = [DNA]
50 is the e estimate for DNA.
Estimate of e for RNA?
Not 50, like DNA. Something like 40.
DNA and RNA are hard to measure directly in a spec because:
They are quite transparent. Absorb UV at 260 nm. Cell wall redirects light, which counts as being absorbed.
Measuring bacteria in a spec:
595-600 nm.
Measuring proteins in a spec:
Absorb UV at 280 nm. AA sequence matters for absorbance - it’s quite sensitive.
Determining DNA purity:
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.
Bradford assay:
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.
Biuret assay:
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.
Compare Bradford and Biuret:
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.
