Extract nucleic acids, run PCR and qPCR, read gels, Western blots and ELISAs, compare sequencing methods, and work with sequences and BLAST.

Molecular Biology Methods

Ladder bands (size in bp, distance from well in mm): 3000 at 9, 2000 at 12, 1500 at 14.5, 1000 at 18, 750 at 22, 500 at 28, 250 at 37, 100 at 46.

Plot log10(size) on the y-axis against distance on the x-axis. Draw a best-fit line through the middle bands (2000 to 250 bp).

Read off the sizes of three unknown bands at 16 mm, 25 mm and 33 mm.

Lane notes: lane 1 ladder, lane 2 NTC (no band), lane 3 positive control (band at 25 mm), lanes 4 to 6 samples (bands at 25 mm, 25 mm and 33 mm plus a faint fuzzy band at 50 mm). The expected product is 600 bp. Write one line of interpretation per lane.

Decide which samples you would report as positive, and what the 33 mm and 50 mm bands might be.

Sketch how you would label the saved gel image (lanes, ladder name and sizes, gel percentage, date, initials).

If your teaching lab runs a gel practical, ask your instructor to show you the lab's gel imaging and image-saving procedure.

## Why it matters

Agarose gel electrophoresis is the quickest way to see DNA. After a PCR, a restriction digest or an extraction, a gel tells you whether you have a product, how big it is, whether there is one band or several, and whether your controls behaved. Reading a gel correctly, and photographing and recording it properly, is a daily skill for biological technicians and an early step in many clinical molecular tests.

## The core idea

DNA has a negatively charged phosphate backbone, so in an electric field it moves toward the **positive electrode (anode)**. A common lab saying is "run to red", because the positive lead is usually red. An agarose gel is a mesh: small fragments slip through quickly, large fragments are slowed. After a run, fragments are sorted by size, smallest farthest from the wells.

To see the DNA, the gel contains (or is soaked in) a fluorescent stain. Many teaching labs use safer stains viewed with blue light; some labs still use ethidium bromide with UV light, which needs specific training and protection because the stain is a mutagen and UV burns skin and eyes. Either way, gel work is done in a lab with your instructor or supervisor, never at home.

## Choosing a gel

| Agarose | Good separation range (roughly) |
|---|---|
| 0.7 percent | about 1,000 to 20,000 bp |
| 1.0 percent | about 500 to 10,000 bp |
| 1.5 percent | about 200 to 3,000 bp |
| 2.0 percent | about 100 to 2,000 bp |

Higher percentages resolve small fragments better. The gel and the tank use the same running buffer (TAE or TBE).

## The ladder

A **DNA ladder** (size marker) is a mix of fragments of known sizes, loaded in its own lane. It is your ruler. Over the useful middle of the gel, the distance a fragment travels is roughly **linear in the logarithm of its size**. So to size an unknown band:

1. Measure the migration distance of each ladder band from the bottom of the well.
2. Plot log10(size in bp) against distance and draw the best straight line through the bands near your unknown.
3. Read the unknown's size from its distance.

**Worked example.** Ladder bands at 1,000 bp (18 mm), 750 bp (22 mm), 500 bp (28 mm) and 250 bp (37 mm). Your PCR band is at 25 mm, between the 750 and 500 bp bands. Interpolating in log space: log10(750) = 2.875 at 22 mm and log10(500) = 2.699 at 28 mm. At 25 mm, halfway, log10(size) is about 2.787, so the size is about **610 bp**. If your predicted product is 600 bp, that is a match. (Simply eyeballing "between 500 and 750" is often enough for a yes/no PCR check.)

## Reading a whole gel

Go lane by lane and write down what you see before you interpret it:

- **Expected lanes:** one sharp band at the predicted size.
- **No-template control:** empty. A band here means contamination.
- **Positive control:** the expected band. If it is missing, the run failed, not your samples.
- **Extra bands:** non-specific products or primer dimers (a faint, fuzzy band below about 100 bp).
- **Smears:** degraded DNA, too much DNA, or too many PCR cycles.
- **Uncut plasmid** can show several bands (supercoiled, nicked, linear) that run at different apparent sizes from the same molecule, which is why you size linear fragments against a linear ladder.

## Recording the result

Save the original gel image without editing it (adjusting overall brightness is usually allowed, editing individual bands never is). Label lanes, the ladder and its sizes, the gel percentage, the date and your initials, and attach the image to your notebook entry. This is the same data integrity you learned for any lab record.

## Common mistakes

- Running the gel backwards, so the DNA runs off the top into the buffer.
- Sizing against the wrong ladder (check which ladder lot you loaded and its band sizes).
- Reading sizes from the far ends of the gel, where the log-linear relationship breaks down.
- Skipping the controls when interpreting the samples.
- Cropping or editing images in ways that hide bands.

## Quick recap

- DNA runs toward the positive electrode; small fragments go farthest.
- Choose agarose percentage by fragment size.
- Size bands by interpolating log10(bp) against distance from a ladder.
- Check NTC and positive control first; record unedited, labelled images.

## Try it

Open the **Do** tab to size bands from a set of gel measurements on paper or graph paper.


Read an agarose gel against a DNA ladder and estimate the size of unknown bands.

Gel electrophoresis

Gel Electrophoresis

EdvoTech Tips: What is a DNA Ladder and how do you use it?

## Why it matters

Gels for DNA tell you about genes. Many questions, though, are about **proteins**: is this protein made, how big is it, and does a treatment raise or lower it? SDS-PAGE separates proteins by size, and a Western blot then detects one specific protein with antibodies. Biochemists, biological technicians and some clinical labs use these methods, and knowing why each step exists is what lets you troubleshoot a bad blot.

## SDS-PAGE: separating proteins by size

Proteins differ in shape and charge, so on their own they would not separate cleanly by size. SDS-PAGE fixes that:

1. **Sample preparation.** Proteins are mixed with a sample buffer containing **SDS** (sodium dodecyl sulfate), a detergent that unfolds proteins and coats them with negative charge roughly in proportion to their length. A **reducing agent** (such as DTT or beta-mercaptoethanol) breaks disulfide bonds. Heating completes denaturation.
2. **Loading.** Equal amounts of total protein (measured beforehand with an assay such as BCA or Bradford) go into each well, plus a **pre-stained protein ladder** of known molecular weights in kilodaltons (kDa).
3. **The gel.** A polyacrylamide gel with a short **stacking gel** on top, which concentrates samples into sharp bands, and a **resolving gel** below, where separation happens. Higher acrylamide percentages resolve smaller proteins.
4. **Running.** Proteins migrate toward the positive electrode; smaller ones move farther.

Afterwards you can stain the whole gel (for example with Coomassie) to see every protein, or move on to a Western blot. Unpolymerised acrylamide is a neurotoxin, which is one reason labs buy precast gels or train people before letting them pour gels.

## Western blot: finding one protein

1. **Transfer.** An electric current moves the proteins out of the gel onto a **membrane** (nitrocellulose or PVDF), keeping their positions. A reversible stain such as Ponceau S confirms the transfer worked.
2. **Blocking.** The membrane is soaked in a protein solution (non-fat milk or BSA) so antibodies do not stick everywhere.
3. **Primary antibody.** An antibody that recognises your target protein binds to it on the membrane.
4. **Washing.** Buffer washes (often TBS or PBS with a little Tween detergent) remove unbound antibody.
5. **Secondary antibody.** An antibody that recognises the primary antibody, linked to an enzyme (such as HRP) or a fluorescent dye. It amplifies and reports the signal.
6. **Washing** again.
7. **Detection.** For HRP, a chemiluminescent substrate produces light captured by an imager; fluorescent secondaries are imaged directly.

## Reading a blot

- **Size check.** Is the band at the expected kDa, judged against the ladder? Modifications such as glycosylation or phosphorylation can shift apparent size.
- **Loading control.** A protein expected to be constant (for example beta-actin, GAPDH or a total-protein stain) shows whether lanes were loaded evenly. Compare the target band to it, never the raw band alone.
- **Controls.** A positive control lysate known to contain the protein, and ideally a negative control (for example a knockdown) that should lack it.
- **Quantification.** Densitometry gives band intensities. Results are semi-quantitative and only valid when the signal is not saturated.

**Worked example.** Target band intensity: control 12,000, treated 6,000. Loading control: control 20,000, treated 10,000. Normalised: control 0.60, treated 0.60. The apparent halving was just uneven loading: there is no real change.

## Troubleshooting by step

| Symptom | Step to check first |
|---|---|
| No bands at all, ladder transferred | Primary antibody, target abundance, detection reagent |
| Ladder also missing from membrane | Transfer |
| Dark background everywhere | Blocking, washing, antibody too concentrated |
| Many extra bands | Antibody specificity, too much antibody, protein degradation |
| Bands smile or smear | Running conditions, overloading, sample prep |

## Common mistakes

- Unequal protein loading, then interpreting band strength without a loading control.
- Saturated signal, which hides real differences.
- Skipping the transfer check.
- Reusing diluted antibody without tracking it.
- Over-editing blot images. Journals and quality systems require original, unedited images.

## Quick recap

- SDS plus reducing agent plus heat: proteins separate by size on polyacrylamide.
- Western: transfer, block, primary, wash, secondary, wash, detect.
- Judge bands against the ladder, controls and a loading control.
- Troubleshoot by asking which step could cause the symptom.

## Try it

The **Do** tab has a quiz on the order and purpose of each step.


Explain what each step of SDS-PAGE and Western blotting does and how to judge a blot.

SDS-PAGE and Western blot

SDS-PAGE explained - Protein Separation Technique

Western Blot / Protein Immunoblot explained

## Why it matters

The enzyme-linked immunosorbent assay (ELISA) measures how much of a specific protein, hormone, antibody or other molecule is in a sample. Research labs use it for cytokines and biomarkers; clinical and public health labs use ELISA-type immunoassays for infections and antibodies; biomanufacturing uses it to measure product and impurities such as host-cell proteins. Running the plate is half the job. The other half is turning optical density readings into concentrations you can defend.

## The core idea: a sandwich ELISA

The most common format for measuring a protein is the **sandwich ELISA** in a 96-well plate:

1. **Capture antibody** is coated on the bottom of each well.
2. **Blocking** with a protein solution covers the remaining plastic.
3. **Sample or standard** is added; the target binds the capture antibody. Washing removes everything else.
4. **Detection antibody**, which binds a different part of the target, is added. It is linked to an enzyme (often HRP), directly or through a biotin and streptavidin step.
5. **Substrate** (often TMB) is added. The enzyme turns it blue; more target means more enzyme and more colour.
6. **Stop solution** (an acid) turns the colour yellow and stops the reaction, and a plate reader measures **optical density (OD)** at 450 nm.

Other formats exist. A **direct** or **indirect** ELISA detects target stuck to the plate; a **competitive** ELISA, used for small molecules, gives *less* signal when there is *more* target.

## The standard curve

OD is not a concentration. You convert it using **standards**: known concentrations of the target, run on the same plate. Typical good practice:

- Run standards, samples and blanks in **duplicate or triplicate**, and use the mean OD.
- **Subtract the blank** (zero standard) OD from everything.
- Fit a curve to the standards. Many ELISAs are sigmoidal over their full range and are fitted with a **four-parameter logistic (4PL)** model in plate-reader software. Over a narrower range the curve is often close to a straight line, which is what this lesson's activity uses.
- **Read unknowns only inside the range of the standards.** A result above the top standard is reported as "above range" and the sample is diluted and rerun; you never extrapolate.

**Worked example.** After blank subtraction, a linear fit of the standards gives slope 0.00208 OD per pg/mL and intercept 0.0 (these standards happen to fall on a perfect line). A sample diluted 1 in 2 reads OD 0.440, or 0.390 after subtracting the blank 0.050.

```text
concentration in well = (0.390 - 0.0) / 0.00208 = 187.5 pg/mL
original sample       = 187.5 x 2 (dilution factor) = 375 pg/mL
```

Forgetting the dilution factor is one of the most common reporting errors.

## Quality checks on a plate

- **Replicate agreement:** the coefficient of variation (CV = SD / mean x 100) of duplicate ODs is often required to be below about 15 to 20 percent. A high CV points to pipetting or washing problems.
- **Curve fit:** check R-squared, and back-calculate each standard from the curve; each should be close to its nominal value.
- **Controls:** kit or in-house controls with known values should fall in their acceptable ranges, just like the QC rules from the lab data course.
- **Edge effects and timing:** uneven temperature or adding substrate slowly across a plate can shift ODs, so follow incubation times exactly.

## Clinical and regulated context

Clinical immunoassays run on validated, regulated systems, and results are released only by qualified staff under CLIA in the US. Medical laboratory scientists qualify through a NAACLS-accredited program (https://www.naacls.org, checked on 2026-10-23) and ASCP Board of Certification exams (https://www.ascp.org/content/board-of-certification, checked on 2026-10-23). Plate work itself happens in a lab, under supervision.

## Common mistakes

- Forgetting to apply the dilution factor.
- Reporting values above the top standard or below the lowest standard as numbers.
- Not subtracting the blank, or subtracting it twice.
- Ignoring high CV between duplicates.
- Mixing standards from one kit lot with plates from another.

## Quick recap

- Sandwich ELISA: capture, block, sample, detection antibody, enzyme, substrate, stop, read OD.
- Convert OD to concentration with a standard curve on the same plate (4PL or, over a linear range, a straight line).
- Blank-correct, stay within the curve, apply dilution factors.
- Check replicate CV, the curve fit and controls before reporting.

## Try it

Open the **Do** tab. A Python script fits the standard curve; you write the line that turns a sample OD into a concentration.


Understand how an ELISA works and compute sample concentrations from its standard curve.

ELISA

ELISA (Enzyme-linked Immunosorbent Assay)

ELISA: How to make a standard curve

Read an agarose gel against a ladder, explain each step of SDS-PAGE and Western blotting, and compute concentrations from an ELISA standard curve.