Biotech Bench

Theory

PCR amplifies a specific DNA region using a heat-stable polymerase (Taq), two primers and a thermal cycler that repeats three temperature steps. Denaturation near 94°C melts the double helix into single strands. Annealing near 55°C lets the short primers bind their complementary sequences. Extension at 72°C is Taq's optimum, where it synthesises the new complementary strand from the primer.

Each complete cycle ideally doubles every target molecule, so after n cycles the copy number is the starting amount times two to the power n. In practice the efficiency E per cycle is below one, and the reaction plateaus once primers, dNTPs or polymerase run low.

Starting from a single molecule, 30 ideal cycles give two to the power thirty, about 1.07 billion copies — the basis of PCR's extraordinary sensitivity.

Procedure

1. Set the starting template copies, the number of cycles and the per-cycle efficiency with the sliders.
2. Press Run program to start the cycler; watch the block temperature trace the 94 / 55 / 72 profile.
3. The reaction tube glows brighter and the amplification chart climbs each cycle on a logarithmic axis.
4. Read the live copy count; note where the curve bends as it approaches the plateau.
5. Lower the efficiency to see fewer copies per cycle, or raise the cycle count to reach saturation.

References

• Mullis, K. B. & Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology 155, 335-350.
• Saiki, R. K. et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491.
• Alberts et al. Molecular Biology of the Cell, 6th ed., Garland Science 2014 — Ch. 8, Analyzing DNA.

Theory

DNA is uniformly negatively charged (phosphate backbone), so in an electric field every fragment is pulled toward the positive anode at a force proportional to its charge. But the agarose gel is a molecular sieve: longer fragments are retarded more by the mesh. The result is that migration distance falls roughly linearly with the logarithm of fragment size — smaller fragments travel farther.

A DNA ladder of known sizes is run alongside the samples; plotting log10(size) against migration distance gives a calibration curve from which any unknown band's size can be read by interpolation.

Procedure

1. Enter fragment sizes (in base pairs) for your sample lane, comma-separated, or use the preset.
2. Set the voltage and press Run to apply the field; bands migrate down their lanes over time.
3. The first lane is a DNA ladder of known sizes for calibration.
4. When the run finishes, click any band to read its estimated size (from the ladder calibration) versus its true size.
5. Raise the voltage to speed migration; note that band order by size never changes.

References

• Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual, 3rd ed., CSHL Press 2001 — Ch. 5, Gel Electrophoresis of DNA.
• Helling, R. B. et al. (1974) Analysis of endonuclease R-EcoRI fragments of DNA from lambda by agarose gel electrophoresis. J. Virology 14, 1235-1244.
• Lee, P. Y. et al. (2012) Agarose gel electrophoresis for the separation of DNA fragments. J. Visualized Experiments 62, e3923.

Theory

Restriction endonucleases are bacterial enzymes that recognise short, usually palindromic DNA sequences and cleave the backbone at a defined position within or near them. EcoRI recognises GAATTC and cuts between G and A, leaving 5' overhangs (sticky ends). Different enzymes have different sites and cut frequencies.

For a linear molecule with k cut sites you obtain k+1 fragments; for a circular plasmid you obtain k fragments. The fragment sizes sum to the total length, and running them on a gel gives a fingerprint used to map and verify constructs.

Procedure

1. Enter or generate a DNA sequence (letters A, C, G, T only).
2. Pick a restriction enzyme; its recognition site and cut offset are shown.
3. Press Digest; every recognition site is highlighted and the molecule is cut.
4. Read the fragment sizes and see them rendered as predicted gel bands.
5. Switch enzymes to compare cut frequencies and fragment patterns.

References

• Smith, H. O. & Wilcox, K. W. (1970) A restriction enzyme from Hemophilus influenzae. J. Molecular Biology 51, 379-391.
• Roberts, R. J. et al. (2015) REBASE — a database for DNA restriction and modification. Nucleic Acids Research 43, D298-D299.
• Watson et al. Molecular Biology of the Gene, 7th ed., Pearson 2014 — restriction mapping.

Theory

The Michaelis-Menten equation describes the initial rate of a single-substrate enzyme reaction. At low substrate the rate rises almost linearly; as substrate saturates the enzyme the rate approaches its maximum, Vmax. The Km is the substrate concentration at which the rate is half of Vmax, an inverse measure of affinity.

The double-reciprocal plot is a straight line: the y-intercept is 1/Vmax and the x-intercept is -1/Km. A competitive inhibitor raises the apparent Km (lines cross on the y-axis, same Vmax); a non-competitive inhibitor lowers the apparent Vmax (lines cross on the x-axis, same Km).

Procedure

1. Set Vmax and Km with the sliders; the hyperbola and Lineweaver-Burk line redraw instantly.
2. Move the [S] marker to read the velocity at any substrate concentration.
3. Choose an inhibitor type and set [I]/Ki to add inhibition; watch the curve shift.
4. Confirm the y-intercept of the double-reciprocal plot gives 1/Vmax and the x-intercept gives -1/Km.
5. Compare how competitive versus non-competitive inhibition changes the apparent parameters.

References

• Michaelis, L. & Menten, M. L. (1913) Die Kinetik der Invertinwirkung. Biochem. Z. 49, 333-369.
• Lineweaver, H. & Burk, D. (1934) The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658-666.
• Nelson & Cox. Lehninger Principles of Biochemistry, 7th ed., W. H. Freeman 2017 — Ch. 6, Enzymes.

Theory

In transcription, RNA polymerase reads the DNA template strand 3' to 5' and builds an mRNA that is complementary to it and identical to the coding strand except that thymine (T) is replaced by uracil (U). In translation, the ribosome reads the mRNA in non-overlapping triplets called codons; each codon specifies one amino acid via the genetic code.

The code is degenerate (64 codons, 20 amino acids) and nearly universal. Translation begins at the first AUG and continues in that reading frame until a stop codon is reached.

Procedure

1. Enter a DNA coding sequence (A, C, G, T) or generate one with a start and stop codon.
2. Press Transcribe to produce the mRNA (T becomes U).
3. Press Translate to read codons from the first AUG and look up each amino acid.
4. The start codon is tagged green and stop codons red; the protein sequence is listed in three-letter and one-letter codes.
5. Edit the sequence to introduce a premature stop and watch the protein truncate.

References

• Crick, F. (1970) Central dogma of molecular biology. Nature 227, 561-563.
• Nirenberg, M. & Leder, P. (1964) RNA codewords and protein synthesis. Science 145, 1399-1407.
• Alberts et al. Molecular Biology of the Cell, 6th ed., Garland Science 2014 — Ch. 6, From DNA to Protein.

Theory

A spectrophotometer passes monochromatic light of intensity I0 through a cuvette of solution and measures the transmitted intensity I. Some light is absorbed by the analyte. Absorbance is the logarithm of the ratio of incident to transmitted light, and by the Beer-Lambert law it is proportional to the molar absorptivity, the concentration and the path length.

Because A is linear in c, a set of known standards gives a straight calibration line A = (ε l) c through the origin. The unknown's concentration is its absorbance divided by the line's slope. The law holds best at low absorbance (below about A = 1).

Procedure

1. Set the molar absorptivity ε and the cuvette path length l.
2. Adjust the sample concentration; the cuvette tints and the detector shows transmittance and absorbance.
3. Press Read standard at several concentrations to plot calibration points.
4. Fit the line, then enter an unknown absorbance to read back its concentration.
5. Push the concentration high and observe the transmitted light fall toward zero as absorbance climbs.

References

• Beer, A. (1852) Bestimmung der Absorption des rothen Lichts in farbigen Fluessigkeiten. Annalen der Physik 162, 78-88.
• Swinehart, D. F. (1962) The Beer-Lambert law. J. Chemical Education 39, 333-335.
• Harris, D. C. Quantitative Chemical Analysis, 9th ed., W. H. Freeman 2016 — Ch. 17, Fundamentals of Spectrophotometry.