Plasmid Extraction Protocol

Materials Provided

• silica gel columns

• LB medium

• 100 mg/ml ampicilin stock (AMP)

• resuspension buffer

• lysis buffer

• neutralization solution

• wash solution

• elution buffer

Step 1

• previously plasmid-containing bacterial cells in LB medium was grown w/ the appropriate antibiotics overnight w/ vigorous agitation

• transfer 1.5ml of bacterial solution to a 1.5ml snap-cap tube

• pellet the cells by centrifuging for 1 min at 13 000 rpm

• decant the supernatant and remove all medium residue by pipet

Step 2

• completely resuspend the cell pellet in 100µl of resuspension buffer by vortexing, vigorous pipetting or drawing across a test tube rack

• no cell clumps should be visible after resuspension of pellet

Step 3

• add 200µl lysis buffer to the resuspended bacteria and gently mix (invert the tube 4-6 times) to lyse the cells

• incubate at room temp for 1 minute

• do not vortex, vortexing will lead to genomic DNA contamination in the plasmid prep

Step 4

• add 350µl neutralisation buffer to neutralize the lysate and gently mix the solution by inverting the tube 3-6 times

• a white precipitate should be formed

Step 5

• centrifuge for 5 mins at 13 000 rpm, meanwhile place a silica column onto a collection tube

Step 6

• transfer 700µl of the supernatant carefully to the column

• centrifuge 1 min at 10 000 rpm

• discard the flow-thru

Step 7

• wash the column once w/ 700µl wash buffer by centrifuging for 1 min at 10 000 rpm

• discard the flow thru

• repeat

Step 8

• centrifuge the column at 13 000 rpm for a further minute to remove residual ethanol

• residual ethanol may inhibit subsequent enzymatic reactions

Step 9

• place the column onto a new 1.5 ml centrifuge tube

• add 50µl elution buffer onto the centre of the membrane

• for effective elution, make sure that the elution buffer is dispensed onto the centre of the membrane and is completely absorbed

Step 10

• stand the column for 2 mins at room temp and centrifuge for 1 min at 13 000 RPM to elute DNA

Step 11

• store plasmid DNA at 4ºC (short term) or -20ºC (longer term)

Theory

Why Cloning instead of PCR?

• PCR can generate large copies of a DNA sequence, but there’s a risk of introducing errors (mutations), which could have consequences for downstream applications

• also, performing further manipulations on the DNA are not possible w/ PCR

  • e.g. wanting to express the bacteria in mammalian cells, simple linear DNA PCR products are not able to express protein in a reliable way

• instead, DNA sequences of interest are introduced into a construct capable of self-replicating within bacteria or other host organisms

Alkaline Lysis

• takes advantage of the physical characteristics of the plasmid to separate it from other biomolecules which make up the host bacteria

• RNAse in the resuspension buffer degrades RNA present in the bacterial lysis and prevents it from contaminating the plasmid prep

• the lysis step, using NaOH and SDS, breaks apart the bacteria, but also denature DNA, meaning that the double-strand structure is converted to two single strand structures

  • this happens to both the plasmid DNA and the bacterial genomes present

• the neutralization quickly returns the pH of the solution near neutral, allowing DNA to renature

• since the plasmid is small, it tends to renature correctly

  • the genome is much bigger and tends to cross-link with other copies of itself and clumps allowing it to precipitate

• the rest of the isolation involves silica gel filtration