P2: This step is alkali treatment. Among them, NaOH is mainly used to dissolve cells and release DNA, because in the case of strong alkalinity, the cell membrane has changed from double-layer membrane structure to microcapsule structure. The purpose of combining SDS with NaOH is to enhance the strong alkalinity of NaOH, and SDS acts as an anionic surfactant to destroy lipid bilayer membrane.
P3: Solution III is used for protein precipitation and neutralization reaction. Among them, potassium acetate forms PDS in order to make potassium ions in SDS replace sodium ions. Because sodium dodecyl sulfate becomes potassium dodecyl sulfate when it meets potassium ions, PDS is insoluble in water, and one SDS molecule binds two amino acids on average, so a large number of precipitates generated by the substitution of potassium and sodium ions naturally precipitate most of protein.
2M acetic acid is used to neutralize sodium hydroxide. Once the genomic DNA is broken, as long as it is a fragment of 50- 100 kb, there is no way to be precipitated by PDS * * * * so the alkali treatment time should be short and violent oscillation is not allowed, otherwise there will always be a large amount of genomic DNA mixed in the finally obtained plasmid, and a thick total DNA band can be observed by agarose electrophoresis.
75% alcohol is mainly used to purify salt and inhibit deoxyribonuclease; ; At the same time, the strong acidity of solution III is also to make DNA better combine with silicate fiber membrane.
Extended data
Plasmids have the ability of self-replication, so that they keep a constant copy number in daughter cells and express the genetic information they carry. Bacterial plasmid is a common vector in DNA recombination technology. Vector is a tool to deliver useful foreign genes into recipient cells for proliferation and expression through genetic engineering.
Recombination of a target gene fragment into plasmid constitutes a recombinant gene or recombinant. Then the recombinant is transformed into recipient cells (such as Escherichia coli) by microbial transformation technology, so that the target gene in the recombinant can be propagated or expressed in recipient bacteria, thus changing the original characteristics of host cells or producing new substances.
Baidu encyclopedia-plasmid
Baidu Encyclopedia-Plasmid Extraction