IIT JAM Biotechnology cheat sheet

Biotechnology - Core Concepts, Techniques and Applications

• Central dogma flow: DNA → RNA → protein; transcription in the nucleus (eukaryotes), translation on ribosomes; one gene–one polypeptide is a simplification because of alternative splicing and polyproteins.

• Restriction enzymes: Type II cut palindromic sequences (e.g., EcoRI GAATTC, BamHI GGATCC, HindIII AAGCTT); sticky ends ligate more efficiently than blunt ends; methylation protects host DNA.

• Cloning vector essentials: origin of replication, selectable marker, multiple cloning site, small size; pBR322 carries ampR and tetR; pUC vectors use ampR plus lacZ α-complementation for blue-white screening.

• PCR cycle: denaturation ~94 °C, annealing ~50–65 °C (≈5 °C below primer Tm), extension ~72 °C with Taq; Tm ≈ 2(A+T) + 4(G+C) °C for short primers; n cycles give ≈2^n copies.

• Gel electrophoresis: DNA migrates toward the anode because phosphate backbone is negatively charged; smaller/supercoiled DNA moves faster; agarose for DNA, polyacrylamide for proteins and small fragments.

• Sanger sequencing uses dideoxynucleotides (ddNTPs) lacking 3'-OH to terminate chain elongation; read length ~500–1000 bp per reaction.

• Southern blot = DNA, Northern blot = RNA, Western blot = protein; detection by labelled probe or antibody; used to confirm presence, size or expression.

• Microbial growth phases: lag, log (exponential), stationary, death; in log phase N = N0 × 2^(t/g), where g is generation time; doubling time g = t/n.

• Sterilisation: autoclave 121 °C at 15 psi for 15–20 min; dry heat 160–180 °C; 0.22 µm filter for heat-labile liquids; UV for surface/air only (poor penetration).