PCR and gel electrophoresis are core laboratory methods used to copy, separate, and analyze DNA. This cheat sheet helps students remember the order of PCR temperature steps, the purpose of each reagent, and how DNA bands move through a gel. It is useful for lab preparation, data analysis, and understanding biotechnology applications such as genetic testing and forensics.
PCR uses repeated cycles of denaturation, annealing, and extension to amplify a target DNA sequence. Gel electrophoresis separates DNA fragments by size because DNA is negatively charged and moves toward the positive electrode. Smaller DNA fragments travel farther through the gel, while larger fragments stay closer to the wells.
Band patterns are interpreted by comparing samples to a DNA ladder or expected fragment sizes.
Key Facts
- PCR stands for polymerase chain reaction, and it makes many copies of a specific DNA region.
- A typical PCR cycle is denaturation at about 95°C, annealing at about 50 to 65°C, and extension at about 72°C.
- Denaturation separates double-stranded DNA into single strands by breaking hydrogen bonds between base pairs.
- Annealing lets primers bind to complementary sequences on the template DNA, which defines the target region to be copied.
- Extension uses DNA polymerase to add nucleotides to the 3' end of each primer and build new DNA strands.
- DNA has a negative charge because of its phosphate backbone, so it moves from the negative electrode toward the positive electrode in a gel.
- Smaller DNA fragments move farther through an agarose gel than larger DNA fragments during the same run time.
- A DNA ladder contains fragments of known sizes, and sample band sizes are estimated by comparing their positions to the ladder bands.
Vocabulary
- PCR
- PCR is a laboratory technique that amplifies a chosen DNA sequence through repeated heating and cooling cycles.
- Primer
- A primer is a short DNA sequence that binds to the template and gives DNA polymerase a starting point for copying.
- DNA polymerase
- DNA polymerase is an enzyme that builds new DNA strands by adding complementary nucleotides.
- Agarose gel
- An agarose gel is a porous material used to separate DNA fragments based on size.
- DNA ladder
- A DNA ladder is a mixture of DNA fragments with known lengths used to estimate the size of unknown bands.
- Band
- A band is a visible line on a gel that represents many DNA fragments of the same or similar length.
Common Mistakes to Avoid
- Putting the gel wells near the positive electrode is wrong because DNA is negatively charged and would run backward out of the wells.
- Forgetting that smaller DNA fragments travel farther is wrong because the gel acts like a sieve that slows larger fragments more.
- Confusing annealing and extension is wrong because primers bind during annealing, while DNA polymerase copies DNA during extension.
- Using the sample band brightness as the main measure of fragment size is wrong because position shows size, while brightness usually relates to DNA amount.
- Interpreting bands without a DNA ladder is unreliable because the ladder provides the known size reference needed to estimate fragment length.
Practice Questions
- 1 A PCR reaction runs for 30 cycles and begins with 1 double-stranded target DNA molecule. Assuming perfect doubling each cycle, how many copies are produced after 30 cycles?
- 2 A gel has bands at 200 bp, 500 bp, and 1000 bp. Which band should be farthest from the wells after electrophoresis?
- 3 A PCR program uses 95°C, 58°C, and 72°C steps. Match each temperature to denaturation, annealing, or extension.
- 4 Explain why a DNA sample moves toward the positive electrode during gel electrophoresis and why fragment size affects how far it travels.