Plasmid preparation
Alkaline lysis of 1–10 mL of overnight culture on a silica spin column, giving roughly 5–30 µg in 30–50 µL and taking about half an hour. The workhorse for screening clones and for routine digests and sequencing.
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Miniprep
Alkaline lysis of 1–10 mL of overnight culture on a silica spin column, giving roughly 5–30 µg in 30–50 µL and taking about half an hour. The workhorse for screening clones and for routine digests and sequencing.
How it differs from the standard method
- Buffer volumes are written for up to about 10 mL of culture; beyond that the lysis is incomplete even though the column could still bind more.
- The column binds about 20–30 µg, which a 10 mL high-copy culture can reach, so for more DNA you move format rather than adding culture.
- Fast enough to do twelve clones in parallel, which is what it is usually for.
1A separation by topology, not by chemistry
A plasmid and the bacterial chromosome are made of the same stuff, so no chemistry can tell them apart. Alkaline lysis separates them by shape instead.
Resuspend
Glucose/Tris/EDTA with RNase A. The cells must be fully resuspended: a clump is never reached by the alkali and never lyses.
Lyse
SDS and NaOH, pH ~12.5. Invert gently 4–6 times until the lysate goes clear and viscous, and keep it to 2–5 min.
Neutralise
Concentrated potassium acetate. Mix immediately: the plasmid reanneals while the chromosome and protein precipitate.
2Why you must not mix vigorously
The whole separation rests on the chromosome being too big to reanneal. Vortexing the alkaline lysate shears it into fragments, and short denatured fragments can find their complements again when the pH drops, so they stay in solution and bind the column alongside your plasmid.
3Yield is set before you start
How much plasmid you can get is decided by three things you choose before touching a kit: the plasmid's origin of replication, how many cells you harvest, and when you harvest them. A pUC-type relaxed-control origin sits at a few hundred copies per cell; a pBR322- or pET-type low-copy origin sits at fifteen to twenty.
4Endotoxin: the prep that clones fine and kills cells
Lipopolysaccharide from the outer membrane of a Gram-negative bacterium is amphipathic and negatively charged, and it co-purifies with plasmid DNA on both silica and anion-exchange resin. A standard miniprep typically carries tens to hundreds of endotoxin units per microgram.
- A260/A230 in a plasmid prep
- The ratio that notices residual chaotropic salt and ethanol from the wash buffer. A skipped dry spin leaves enough of both to inhibit restriction enzymes and ligase, and it shows here long before it shows in A260/A280.
- Residual RNA
- The commonest reason an absorbance concentration is wrong. RNA's own A260/A280 is about 2.05 against DNA's 1.88, so a prep can carry as much RNA as plasmid and move the ratio by less than 0.1, which is why a dsDNA-specific dye is the right tool when the number has to be right.
Key terms
Practise these as flashcards →The SDS/NaOH then potassium acetate method that separates plasmid from chromosome by whether the denatured DNA can reanneal on neutralisation.
Plasmid molecules per cell, set by the origin of replication: a few hundred for relaxed-control pUC-type origins, fifteen to twenty for stringent low-copy ones.
The negatively supercoiled, covalently closed form native to a bacterial plasmid. It is compact, runs fast on a gel, and transfects better than relaxed or nicked DNA.
A plasmid with a nick in one strand, so the supercoiling has relaxed. It still digests and sequences, but it migrates more slowly and transfects less well.
Covalently closed DNA that spent too long in alkali and could not pair correctly again. It runs ahead of supercoiled DNA, resists restriction enzymes and is useless for transfection.
The mass of DNA a column's resin can hold, roughly 20–30 µg for a mini, 100–120 µg for a midi, 500–600 µg for a maxi. Plasmid beyond it is lost.
The activity measure for lipopolysaccharide, assayed by LAL. Standard preps run at tens to hundreds of EU/µg; endotoxin-free preps are below 0.1.
The supernatant after the neutralised lysate has been chilled and spun hard, or filtered. Loading it before the floc has compacted is what clogs columns.
When to use it
- Screening transformants: a miniprep from a few millilitres gives enough DNA for a diagnostic digest and a sequencing reaction.
- Making a working stock of a verified construct to digest, subclone or sequence from over the following months.
- Producing tens to hundreds of micrograms for transfection, in vitro transcription or large-scale digests, where a midi or maxi format is needed.
- Preparing DNA for primary cells, stem cells, immune cells or in vivo work, where endotoxin content decides whether the experiment works at all.
Compared with alternatives
Prefer it when the target is the chromosome rather than the plasmid; it cannot use the topology trick, so it needs proteinase K and a capture chemistry instead.
How you check the prep: a dsDNA dye for the concentration you will actually rely on, absorbance ratios for carry-over, and a gel for the mix of plasmid forms.
The step before: it puts the plasmid into the cells whose overnight culture you are about to harvest.
The application that makes endotoxin and supercoiling matter, and the reason endotoxin-free preps exist.
The quickest way to see what is in a prep: supercoiled, open-circle and denatured plasmid all migrate differently, and chromosomal carry-over shows as a high haze.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Alkaline lysis of 1–10 mL of overnight culture on a silica spin column, giving roughly 5–30 µg in 30–50 µL and taking about half an hour. The workhorse for screening clones and for routine digests and sequencing.
- Buffer volumes are written for up to about 10 mL of culture; beyond that the lysis is incomplete even though the column could still bind more.
- The column binds about 20–30 µg, which a 10 mL high-copy culture can reach, so for more DNA you move format rather than adding culture.
- Fast enough to do twelve clones in parallel, which is what it is usually for.