Covers the four basic molecular biology methods in turn: gel electrophoresis separating DNA by size, with ethidium bromide staining and UV visualisation, then PCR with its primers, reagents and denaturation, annealing and extension cycle, and RT-PCR for RNA via cDNA. It goes on to Southern, Northern and Western blotting, then Sanger and next-generation sequencing. A set of short mnemonics summarising each method is included.
Topic 42 of 105 in the MU Sofia syllabus for Biology (Medicine, Year 1). The free AceMed note covers it in 7 sections, about 1,051 words (7 min of reading), with 13 flashcards.
In this note
Basic methods in molecular biology - electrophoresis, PCR, blotting, sequencing.
Electrophoresis
Polymerase Chain Reaction (PCR)
Reverse Transcription PCR (RT-PCR)
Blotting Techniques
DNA Sequencing
MNEMONICS
Electrophoresis
Electrophoresis is a technique used to separate DNA fragments based on their size by applying an electric field.
Key Principle
Charged molecules, like DNA, move when exposed to an electric field.
DNA is negatively charged due to its phosphate backbone and migrates towards the positively charged electrode (anode).
Method Overview
DNA samples are loaded into wells within a gel matrix, often made of agarose.
An electric current is passed through the gel.
Smaller DNA fragments move more quickly through the gel, while larger fragments move more slowly, allowing for size-based separation.
Visualizing the Results
After the run is complete, the gel is stained with a fluorescent dye(Ethidium bromide) that binds to DNA.
Under ultraviolet light, the DNA fragments appear as bright bands.
The position and intensity of these bands can be compared to molecular weight markers to assess fragment size and concentration.
Use in PCR Analysis
Electrophoresis is commonly used to verify the success of a PCR reaction.
Following PCR, the product is run on a gel. If amplification was successful, a visible band will appear at the expected size.
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