Biology

Molecular lab sketch

Restriction enzymes, electrophoresis, PCR, and sequencing — a short principle sketch.

Basics

Restriction enzymes and plasmids

Restriction enzymes cut DNA at short recognition sites. Sticky ends leave overhangs that anneal to matching pieces; blunt ends are harder to join. A plasmid is a small circular bacterial DNA with an origin and often an antibiotic marker, used as a cloning vector. Ligase seals the phosphodiester backbone. This is a study sketch, not a protocol.

Gel electrophoresis

DNA is negative, so it runs toward the anode. In an agarose mesh, short fragments move faster. Dyes and UV (or blue light) show bands against a size ladder. Proteins are separated near their mass on SDS–PAGE. Buffer pH and voltage change resolution. A band says presence and rough size, not the full sequence.

PCR

The polymerase chain reaction repeats denaturation (strand separation), annealing (primers), and extension (a heat-stable polymerase) for dozens of cycles so a target grows exponentially. Primers set specificity; cycle count and enzyme set yield. qPCR watches each cycle with fluorescence; RT-PCR reverse-transcribes RNA to cDNA first. One contaminated tube can invent a false positive.

Sequencing and editing sketch

Sanger sequencing uses chain-terminating nucleotides to make length ladders read by colour. Short-read mass sequencing assembles fragments or calls variants. CRISPR–Cas is a sketch of a tool that cuts where a guide RNA points. This is classroom language, not guidance for therapy or environmental release. Always read a result beside a negative control.

Formulas

Ideal PCR amplification

N = N₀ (1 + E)^n

If efficiency E=1, doubles each cycle. Real runs are lower.

Symbols

N₀ starting template count
E amplification efficiency (0–1)
n cycle count

Primer Tm (rough)

T_m ≈ 4(G+C) + 2(A+T) (°C, 짧은 프라이머)

Wallace rule. Long primers and salt need other formulae.

Symbols

G+C count of G and C
A+T count of A and T

Electrophoretic mobility (sketch)

v ∝ q E / f

Charge q, field E, drag f (size and gel).

Symbols

v drift speed
f friction factor

Key table

Taq	polymerase from Thermus aquaticus; survives denaturation heat
EtBr vs safer dyes	ethidium intercalates and is mutagenic; classrooms prefer safer dyes
Kozak / Shine–Dalgarno	sequence near translation start; eukaryote vs bacterium