Molecular Biology protocols

Introduce a defined single-nucleotide substitution (e.g., a missense mutation) into a target gene on a plasmid using complementary mutagenic primers, whole-plasmid PCR, DpnI removal of template, and sequence-verified clone recovery.

Seamlessly join a PCR-amplified gene insert into a linearized expression vector using Gibson/isothermal assembly, then transform, screen, and sequence-verify clones to obtain a correct, in-frame construct.

Quantify relative mRNA expression of a target gene across treatment conditions using two-step SYBR Green RT-qPCR, normalized to two experimentally validated reference genes, with MIQE-compliant controls and ΔΔCq analysis.

Isolate high-integrity, DNA-free total RNA (RIN ≥ 8) from cultured adherent mammalian cells suitable for downstream RT-qPCR and RNA-seq, with quantitative QC of yield, purity, and integrity.

Clone a PCR-amplified insert into an expression vector using two distinct restriction sites for directional, ligation-based insertion, with dephosphorylation of the vector to suppress self-ligation and blue/white or PCR screening of recombinants.