PCR cycles denaturation (95 C), primer annealing (55-65 C), and extension by heat-stable Taq polymerase (72 C). Amplification is exponential: N=N0×2n so 30 cycles yield about copies. Taq lacks proofreading; Pfu is the high-fidelity choice. RT-PCR first converts mRNA to cDNA; qPCR quantifies in real time.
PCR, electrophoresis, blotting, and gene editing tools
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Gel electrophoresis runs negatively charged DNA toward the anode; smaller fragments migrate farther.
Blots, Cutting, and Cloning
SNoW DRoP: Southern detects DNA, Northern RNA, Western protein (antibody probe).
Restriction enzymes cut palindromic sites, leaving sticky or blunt ends. Cloning: cut insert and vector with the same enzyme, ligate, transform bacteria, select on antibiotic, then screen. Express human genes from cDNA — bacteria have no spliceosome to remove introns.
Editing and Sequencing
CRISPR-Cas9 uses a guide RNA to direct a double-strand break: NHEJ repair yields knockouts via indels; homology-directed repair with a donor template gives precise edits.
Sanger sequencing exploits ddNTPs that lack a 3'-OH, terminating chains at every position; next-generation sequencing runs massively parallel short reads. FISH localizes sequences on chromosomes; flow cytometry sorts labeled cells.
Key Takeaways
PCR needs template, two primers, dNTPs, Taq, and magnesium.
Match each blot to its molecule and probe.
cDNA plus a selectable marker is the core recombinant-protein strategy.
CRISPR outcome depends on which repair pathway fixes the cut.