Experiment – 2

Plasmid DNA Isolation using Alkaline Lysis Method

 Plasmids
• Small, circular DNA molecules which many bacteria possess in
addition to having chromosomal DNA.
• Present in many copies per cell, whereas others are present in only
one or two copies.
• Carry genes that are not essential to bacterial function but that may
play an important role in the life cycle and growth of their bacterial
hosts.
• Extensively in genetic engineering and some of them play a role in
the spread of antibiotic resistance among bacteria
 Plasmids
• Circular and several thousand base pairs
in length
• Possess its own origin of replication
• Replicates independently of the bacterial
chromosome
• Replication proceeds from the origin in
one or two directions until the entire
plasmid is copied
• Few plasmids have multiple replication
origins
Supercoiled Plasmid
• Native confirmation found in vivo and occurs when extra twists are introduced into the
double helix strand.
• Supercoiled DNA migrates faster than predicted in an agarose gel due to its conformation.
• Supercoiled DNA is the desired species when isolating plasmid DNA.
Nicked, Relaxed Circular Plasmid
• DNA found in the supercoiled form is not easily accessed by replication machinery.
• During replication, cellular topoisomerases nick one strand of the DNA helix and relax the
superhelical tension, thus allowing polymerases to gain access to the DNA.
• Slowest migrating form in an agarose gel.
• Linear Plasmid
Linearized DNA occurs when the DNA helix is cut in both strands at the same place.
Migrates between the nicked circle and the supercoiled forms.
• Circular, Single Stranded Plasmid
When the alkaline lysis step is overly harsh (e.g. it is incubated for too long) the DNA can
become permanently denatured and gives a single stranded closed circles that migrate
ahead of all of the other forms of the plasmid in a gel.
 Goal

• Isolate extra-chromosomal, plasmid DNA from bacterial cells that

contain the plasmid.
• Method used is alkaline lysis method.
• The various steps involved are:
1. Re-suspension
2. Lysis
3. Neutralization
4. Cleaning and concentration
• Solution I - 25mM Tris HCl pH 8.0
10mM EDTA pH 8.0
50mM Glucose
• Solution II - (500µl) – Add 20µl of 5N NaOH
50µl 10% SDS
430µl of sterile water.
Mix by inverting.
• Solution III - 3M Sodium Acetate pH 5.2
 Resuspension
• Growth of the bacterial cell culture harboring desired plasmid with a
selection marker.
• When sufficient growth has been achieved, the cells are pelleted by
centrifugation to remove them from the growth medium
• Pellet is then re-suspended in a solution containing:
• Tris – EDTA
- Chelates divalent cations in the solution preventing DNases from damaging the plasmid and
also helps by destabilizing the cell wall.
• Glucose
• -Glucose maintains the osmotic pressure so the cells don’t burst
• RNase A
• - Degrades cellular RNA when the cells are lysed
• Divalent cations (Mg2+, Ca2+)
• Essential for DNase activity
• Integrity of the bacterial cell wall
 Lysis
Lysis buffer ( Solution II)
• Sodium hydroxide (NaOH)
 Helps to break down the cell wall
 Disrupts the hydrogen bonding between the DNA bases
 Converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and
plasmid, to single stranded DNA (ssDNA).
 Process is called denaturation and is central part of the procedure, which is why it’s called alkaline lysis
• Detergent Sodium Dodecyl (lauryl) Sulfate (SDS)
• Solubilizes the cell membrane.
• Denatures most of the proteins in the cells, which helps with the separation of the proteins from the
plasmid later in the process.
Neutralization
• Addition of Potassium Acetate (solution III)
• Decreases the alkalinity of the mixture.
• Hydrogen bonding between the bases of the single stranded DNA can be re-
established
• ssDNA can re-nature to dsDNA (Selective part)
• Easy for small circular plasmid DNA to re-nature
• Impossible to properly anneal huge gDNA stretches.
• It’s important to be gentle during the lysis step because vigorous mixing or
vortexing will shear the gDNA producing shorter stretches that can re-anneal and
contaminate plasmid prep.
• While the double-stranded plasmid can dissolve easily in solution, the single
stranded genomic DNA, SDS and the denatured cellular proteins stick together
through hydrophobic interactions to form a white precipitate.
• The precipitate can easily be separated from the plasmid DNA solution by
centrifugation
Cleaning and concentration
Cleaning up the solution and concentrate the plasmid DNA
Including phenol/chloroform extraction followed by ethanol
precipitation
Affinity chromotography-based methods.
Source: [Link]
1) Take bacterial cell pellet (obtained from 1ml culture of [Link]).
2) Resuspend the pellet in 100µl of solution I by vortexing at RT.
3) Add 200µl of solution II to resuspended pellet. Mix the contents by inverting the tube 5 to 10 times. Do not vortex. Store on ice.
4) A slightly viscous and transparent solution will be obtained.
5) Incubate for 2min at RT.
6) Add 150µl of Solution III.
7) Mix by gently inverting to obtain a white flocculent precipitate and then place the tube on ice for 5 min.
8) Spin at 12,000 g for 5 min in an eppendorf.
9) Carefully transfer the supernatant to another 1.5 ml tube
10) Add 450µl of DNA precipitation solution to the supernatant, mix by inverting and leave at RT for 10 min.
11) Spin at 12,000g for 5 min.
12) Discard the supernatant
13) Add 500µl of wash solution to the pellet, vortex to wash the pellet, and centrifuge at 12,000 g for 5 min.
14) Discard supernatant
15) Air dry the pellet for 5 to 10 min. Donot over dry the pellet
16) Resuspend the pellet in TE Buffer.
17) Run on 1.2 % agaraose gel.
 Precautions

1. Complete Lysis (step 2) and neutralization( step 3) within 3 min.

2. Carefully discard the supernatant with dislocating the pellet.
3. Donot over dry the pellet; since an overdried pellet will be difficult
to re-suspend.