Lab Skills Assessment Review

On a P20 micropipette (range 0.5-20 microL) what place value does each digit in the 3-digit readout represent from top to bottom?

Top digit = Tens (microL) Middle digit = Units (microL) Bottom digit = Tenths (microL)

On a P200 micropipette (range 20-200 microL what place value does each digit in the 3-digit readout represent from top to bottom?

Top digit = Hundreds (microL) Middle digit = Tens (microL) Bottom digit = Units (microL)

On a P1000 micropipette (range 100-1000 microL), what place value does each digit in the 3-digit readout represent from top to bottom?

Top digit = Thousands (microL) Middle digit = Hundreds (microL) Bottom digit = Tens (microL)

What volume is displayed on a P20 micropipette with the readout [ 1 ] [ 4 ] [ 5 ]?

14.5 (10 + 4 + 0.5) microL

What volume is displayed on a P20 micropipette with the readout [ 0 ] [ 7 ] [ 2 ]?

7.2 (0 + 7 + 0.2) microL

What volume is displayed on a P20 micropipette set to its maximum capacity with the readout [ 2 ] [ 0 ] [ 0 ]?

20.0 (20 + 0 + 0.0) microL

What volume is displayed on a P20 micropipette with the readout [ 0 ] [ 0 ] [ 8 ]?

0.8 (0 + 0 + 0.8) microL

What volume is displayed on a P20 micropipette with the readout [ 1 ] [ 0 ] [ 0 ]?

10.0 (10 + 0 + 0) microL

What volume is displayed on a P200 micropipette with the readout [ 1 ] [ 7 ] [ 5 ]?

175 (100 + 70 + 5) microL

What volume is displayed on a P200 micropipette with the readout [ 0 ] [ 4 ] [ 2 ]?

42 (0 + 40 + 2) microL

What volume is displayed on a P200 micropipette set to its maximum capacity with the readout [ 2 ] [ 0 ] [ 0 ]?

200 (200 + 0 + 0) microL

What volume is displayed on a P200 micropipette with the readout [ 0 ] [ 9 ] [ 6 ]?

96 (0 + 90 + 6) microL

What volume is displayed on a P200 micropipette with the readout [ 1 ] [ 3 ] [ 0 ]?

130 (100 + 30 + 0) microL

What volume is displayed on a P1000 micropipette with the readout [ 0 ] [ 6 ] [ 5 ]?

650 (0 + 600 + 50) microL

What volume is displayed on a P1000 micropipette set to its maximum capacity with the readout [ 1 ] [ 0 ] [ 0 ]?

1000 (10 + 0 + 0) microL (equivalent to one mL)

What volume is displayed on a P1000 micropipette with the readout [ 0 ] [ 2 ] [ 0 ]?

200 (0 + 200 + 0) microL

What volume is displayed on a P1000 micropipette with the readout [ 0 ] [ 8 ] [ 2 ]?

820 (0 + 800 + 20) microL

What volume is displayed on a P1000 micropipette with the readout [ 0 ] [ 4 ] [ 7 ]?

470 (0 + 400 + 70) microL

In experimental design, what is a factor?

A condition or independent variable that is manipulated or changed in an experiment.

What does the term 'levels' refer to when describing an experimental factor?

The number of different values, strengths, or forms of a particular factor tested (including control treatments such as 0 microM.

What is a treatment in an experimental setup?

A specific combination of various levels of factors applied to experimental subjects.

What is a dependent variable?

The response variable measured to quantify the effect of the manipulated factor(s).

What are controlled variables?

Factors that could influence the dependent variable but are explicitly held constant across all replicates and treatments.

How do experimental replicates differ from technical replicates?

Experimental replicates involve measuring separate subjects or samples to capture biological variation. Technical replicates involve repeated measurements of the exact same sample to assess measurement precision.

What is pseudo-replication?

Treating repeated measurements on a single experimental subject as independent experimental replicates.

What role does randomization play in experimental design?

It uses unbiased assignment methods to allocate treatments to replicates, preventing systematic bias.

How are research questions, hypotheses, and predictions related?

A research question states what is being investigated. A hypothesis proposes a tentative answer. A prediction extends the hypothesis to a specific, measurable experimental outcome.

What is a null hypothesis (H0) in statistical analysis?

The default statistical assumption that no real difference or effect exists between comparison groups.

What parameter is calculated by a t-test to compare two sample means?

The test statistic $t$, representing the ratio of variation between groups to variation within groups.

When is a 1-way ANOVA used instead of a t-test?

When evaluating differences among the means of three or more treatment groups for a single independent variable.

Why is running a 1-way ANOVA preferred over conducting multiple pairwise t-tests?

Performing multiple t-tests accumulates Type I error risk across comparisons, whereas an ANOVA controls the overall error rate.

What does a significant p-value (p < 0.05) from a 1-way ANOVA indicate?

At least one group mean is significantly different from the others, requiring a post-hoc test to identify specific pairwise differences.

What is the purpose of a post-hoc test (e.g., Tukey HSD) following an ANOVA?

To perform specific pairwise comparisons between individual treatment means to determine which groups differ significantly.

Graph of Number of Taxa versus Habitat with ANOVA p=0.01 and post-hoc letters A over Forest, A over Grassland, and B over Urban. Based on the post-hoc letter labels (A, A, B) in this plot, what can be concluded about the mean number of taxa across habitats?

Forest and Grassland (both labeled 'A') do not differ significantly from each other, but both have a significantly higher mean number of taxa than Urban (labeled 'B').

How many test statistics (F-values) are calculated in a 2-way ANOVA?

Three separate F-values: one for Factor 1, one for Factor 2, and one for the interaction between Factor 1 and Factor 2.

In R, what is the purpose of the Tidyverse pipe command %>%?

It passes ('pipes') the output of one function directly into the first argument of the next function (read as 'and then').

State the R code block that correctly calculates the mean and standard deviation of dry_biomass grouped by habitat?

descriptive_stats

In R, which function is used to fit a 1-way ANOVA model, and which function runs a post-hoc test on that model?

ANOVA model: aov() Post-hoc test: TukeyHSD()

What is the function of Phosphate Buffered Saline (PBS) during tissue preparation in DNA isolation?

It acts as a buffer to maintain a consistent pH while mechanical grinding disrupts the cell structure.

What active reagents are present in Qiagen Buffer AL, and what are their functions?

Guanidine hydrochloride (a chaotropic salt) breaks cell membranes and disrupts hydrogen bonding to release cellular DNA into solution.

Why is Proteinase K added during cell lysis in genomic DNA isolation?

It degrades cellular proteins, specifically digesting endonucleases/DNases that would otherwise hydrolyze target DNA.

Why is 100% Ethanol added to the cell lysate prior to loading onto a silica spin column?

In high-salt conditions, ethanol precipitates nucleic acids, facilitating selective binding of DNA to the silica filter.

How does Buffer AW1 differ from Buffer AW2 in silica column DNA purification?

Buffer AW1 contains guanidine hydrochloride and ethanol to wash away cellular contaminants while keeping DNA bound. Buffer AW2 lacks chaotropic salt to remove residual salts prior to elution.

Why must waste containing guanidine hydrochloride never be mixed with bleach?

Combining guanidine hydrochloride with sodium hypochlorite (bleach) generates toxic chlorine gas.

How is purified DNA eluted off the silica filter in the final step of isolation?

Warm (37 degree C) dH2O or elution buffer is added to solubilize DNA off the silica matrix, followed by centrifugation into a clean microtube.

Graph showing DNA yield versus Amount of starting material. The actual yield curve peaks near the Maximum starting material threshold and then declines. Why does using starting tissue beyond the recommended maximum threshold reduce actual DNA yield?

Excessive tissue leads to incomplete lysis and overloads the silica matrix, clogging the membrane and reducing total DNA recovery.

In molecular biology protocols, what is the difference between a pellet and a supernatant?

The pellet is the solid mass collected at the bottom of a tube after centrifugation. The supernatant is the clear liquid remaining above the pellet.

In what standard directional orientation must nucleotide primer sequences always be written?

5' to 3' direction.

Given a DNA coding strand running 5' to 3', how is the forward PCR primer sequence derived?

It is identical to the 5' end sequence of the target region, written in the 5' to 3' direction.

Given a DNA coding strand running 5' to 3', how is the reverse PCR primer sequence derived?

It is the reverse complement of the 3' end sequence of the target region, written 5' to 3'.

For the DNA sequence 5'- cctaaccgtt gcacccttcc attggtatgc cttcgctacg tttcttgaac -3', what is a 10 bp forward primer?

5'- cctaaccgtt -3'

For the DNA sequence 5'- cctaaccgtt gcacccttcc attggtatgc cttcgctacg tttcttgaac -3', what is a 10 bp reverse primer written 5' to 3'?

5'- gttcaagaac -3' (the reverse complement of 3'- caagaacttg -5').

If a PCR amplicon spans nucleotide position 1490 through nucleotide position 2198, what is the expected size of the PCR product?

(2198 - 1490 + 1) = 709 bp

What structural modification distinguishes a dideoxynucleotide (ddNTP) from a standard deoxyribonucleotide (dNTP)?

A ddNTP lacks a hydroxyl group (-OH) at the 3' position of the deoxyribose sugar.

Why does incorporating a dideoxynucleotide (ddNTP) halt DNA chain extension during Sanger sequencing?

Without a 3'-OH group, DNA polymerase cannot form a phosphodiester bond to add the next incoming nucleotide.

In fluorescent Sanger sequencing, how are chain-terminating nucleotides distinguished?

Each of the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) is tagged with a distinct fluorescent dye.

How are DNA extension fragments separated by size in automated Sanger sequencing?

Capillary Electrophoresis (CE) using a glass capillary filled with a gel polymer.

What is the relationship between DNA fragment migration speed during Capillary Electrophoresis and its molecular weight?

The migration speed is inversely proportional to molecular weight (smaller fragments migrate faster).

How does an automated capillary sequencer detect terminated DNA fragments as they migrate?

A laser excites the fluorophore at the capillary window, and a light sensor records the wavelength.

What do the individual colored peaks on a Sanger sequencing electropherogram represent?

Each peak represents fluorescent emission from a dye-labeled ddNTP terminating a fragment at that base position.

What is the function of Sodium Dodecyl Sulfate (SDS) in SDS-PAGE?

SDS denatures proteins by breaking non-covalent bonds and coats them with a uniform negative charge density proportional to mass.

Why are reducing agents like DTT or beta-mercaptoethanol (BME) included in SDS-PAGE sample buffer?

To reduce and cleave covalent disulfide bonds between cysteine residues, linearizing tertiary and quaternary structures.

What is the role of heating protein samples to 95 degrees Celsius prior to SDS-PAGE loading?

It speeds up protein denaturation and enhances the binding of SDS and reducing agents.

In SDS-PAGE, what is the functional difference between the stacking gel and the resolving gel?

The low-density stacking gel focuses all proteins into tight, sharp bands. The denser resolving gel separates proteins by molecular weight.

Diagram of vertical SDS-PAGE apparatus showing sample loading, cathode (-), anode (+), porous gel matrix, and direction of anion migration. During SDS-PAGE, toward which electrode do SDS-coated proteins migrate?

Toward the positively charged anode (+) electrode, driven by the negative charges imparted by SDS.

In Western blotting, what role is played by the primary antibody?

It binds specifically to the target protein antigen immobilized on the membrane.

In Western blotting, what role is played by the secondary antibody?

It binds specifically to the primary antibody and carries a conjugated reporter enzyme.

What is the function of a blocking protein (e.g., non-fat dry milk or BSA) in Western blotting?

It binds non-specifically to open sites on the membrane, preventing background antibody binding.

What is the role of a conjugated enzyme on the secondary antibody in Western blotting?

It catalyzes a chemical substrate reaction producing a visual or chemiluminescent signal that marks target protein bands.