PCR and Gel Electrophoresis

PCR: a copy machine for DNA

Cloning in bacteria is powerful but slow. The polymerase chain reaction (PCR) copies a specific DNA segment in a test tube, with no cells at all, in a couple of hours. It borrows the cell's own copying enzyme, DNA polymerase, and repeats the same three-step cycle over and over.

What makes PCR copy only the stretch you want? Short, custom-made DNA pieces called primers. You design two primers that base-pair to the sequences flanking your target — one on each strand. The polymerase only starts copying from a primer, so it fills in exactly the region between them and nothing else.

1. Denature: heat to ~95 °C so the double helix unzips into two single strands.
2. Anneal: cool to ~55 °C so the primers base-pair to their matching sites.
3. Extend: warm to ~72 °C so DNA polymerase builds new strands from the primers.
4. Repeat the cycle 25–35 times; each round doubles the target, so copies grow exponentially.

Gel electrophoresis: sorting by size

Once you have DNA pieces, you need to see them and check their sizes. Gel electrophoresis does this. You load DNA into wells at one end of a slab of jelly-like gel and apply an electric field. DNA is negatively charged, so it migrates toward the positive electrode — but it has to wriggle through the gel's mesh.

Smaller fragments slip through the mesh easily and travel far; larger fragments drag and stay near the wells. After a while the mixture spreads into separate bands, sorted by size. Comparing your bands to a 'ladder' of known sizes tells you how big each piece is — a quick check that PCR or a restriction cut gave you the fragment you expected.

Gel after the run (DNA moves DOWN, toward + electrode):

   wells:  [Ladder] [Sample]      <- start, near - electrode
           =====    =====
   large   --3000--                slow, stays high
           --2000-- ----2000----   sample band lines up at 2000
   small   --1000--                fast, travels far
                    ---- (none) ----
           v v v v  v v v v
                                    <- finish, near + electrode

Reading: the sample's single band sits level with the 1000-bp
rung of the ladder, so the PCR product is about 1000 base pairs.