BIOL155 Lab: Isolation of Plasmid DNA a.k.a The Mini-Prep
Introduction:
The DNA of prokaryotic cells is relatively simple in comparison to the DNA of eukaryotic cells. A bacterium has
about 1/1000 as much DNA as a eukaryotic cell. In addition to chromosomal DNA, a bacterium may also carry
an additional circular piece of DNA called a plasmid. Plasmids are useful to bacterial cells because they can carry
genes (such as antibiotic resistance) that allow them to grow in a non-ideal environment. The plasmid has
proven to be a useful tool for the molecular biologist. Genes can be inserted into the plasmid which in turn can
be incorporated into a bacterium by a process termed transformation. Since bacteria will rapidly multiply and
create many copies of the gene, the molecular biologist can retrieve relatively large quantities of a protein
produced by the bacteria or can use this arrangement to study the function of a particular gene.
The mini-prep is a quick method for isolating small amounts of plasmid DNA (~1μg/mL of culture) from a
transformed host. The mini-prep is convenient and not labour intensive and applicable to cloning experiments
where it allows rapid screening of transformants. Various methods have been developed for rapid small-scale
plasmid isolation. The most notable are the alkaline lysis method (Birnboim and Doly 1979) and the rapid
boiling procedure (Holmes and Quigley 1981). Several rapid methods for the isolation of plasmid DNA based
on chromatographic purification are now commercially available and widely used (e.g. QIAgen, Invitrogen).
In the alkaline lysis method, alkaline SDS lysis of the bacteria frees the plasmid from the E. coli host leaving
behind the larger chromosomal DNA with the lysed debris that is subsequently cleared by a brief
centrifugation. If necessary, RNA can be digested away with the enzyme RNAse A if it may impede
downstream experiments. Generally, this is not necessary as the contaminating RNA is not a problem in many
procedures and furthermore there are many RNases around (on glassware, your skin etc.) that result in the
degradation of RNA unless you are extremely careful to avoid it. The plasmid DNA is further purified from
proteins by phenol: chloroform extraction and concentrated by ethanol precipitation.
In this lab you will use the QIAgen Spin Miniprep kit that is designed for quick, convenient processing of 1-24
samples simultaneously in less than 30 minutes. This particular protocol is designed for purification of up to
20μg of high-copy plasmid DNA from 1-25mL overnight cultures of E. coli grown in LB (Luria-Bertani) medium.
It is based on the modified alkaline lysis method of Birnboim and Doly.
Remember to think about what is actually occurring in each step!!
While working with DNA it is extremely important not to expose the DNA to DNAse that is present on your
hands, unautoclaved reagents, pipette tips and glassware. In order to eliminate contamination and
destruction of your DNA sample by DNAse, gloves must be worn when working with DNA. In addition, all
reagents and tips that touch DNA solutions or any reagents that will be added to them must be autoclaved.
After these items have been sterilized the proper techniques must be used to maintain their sterility.
Learning Objectives:
• Practice preparing plasmid DNA
• Practice working with small volumes
• Practice working with DNA
• Become familiar with kit column commonly used for plasmid isolation
• Dispose of waste appropriately
Qiagen Spin Miniprep Kit Protocol (Per Student):
*Note all biohazardous waste material e.g. tips, tubes etc. should be disposed of in the appropriate
biohazard containers.
A culture containing the pAMP plasmid has been inoculated into LB (Luria-Bertani) broth and incubated
overnight with shaking at 37°C.
1a. Using a high-speed microcentrifuge (at max), spin down all of your culture to collect a dense cell pellet
To do this: Fill a 1.5mL eppie using a sterile transfer pipette, spin for ~30sec-1min @ max and remove
supernatant (you can pour it off into the biohazards waste). Continue doing this until you have used all of
your o/n culture. *You can rest the transfer pipette in your broth tube between spins.
1b. Resuspend cell pellet in 250μL of Buffer P1
No cell clumps should be visible after resuspension of the pellet. You can use a wide-bore pipette (P-1000 or
transfer pipette) to pipette up and down until your pellet is fully in solution.
2. *Note: Buffer P2 contains NaOH and can be harmful
Add 250μL of Buffer P2 and invert the tube 4–6 times to mix
This buffer is a NaOH/SDS buffer which results in the lysis and release of the cell contents and denaturing and
release of the DNA and proteins. Optimized lysis time allows maximum release of plasmid DNA without
chromosomal DNA.
Mix gently by inverting the tube. Do not machine vortex, as this will result in shearing of genomic DNA
(gDNA). If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. *Do not
allow the lysis reaction to proceed for more than 5min. otherwise your plasmid DNA will also denature and
you will not be able to separate it out from your chromosomal DNA.
3. *Note: N3 contains guanidine hydrochloride (chaotropic) and acetic acid (corrosive) and can be harmful
Add 350μL of Buffer N3 and invert the tube immediately but gently 4–6 times. The lysate is neutralized by
N3 and adjusted to high salt conditions that result in denaturing chromosomal DNA and proteins but not
smaller plasmids. *To avoid localized precipitation, mix the solution gently but thoroughly, immediately after
addition of Buffer N3. The solution should become cloudy.
4. Centrifuge for 10min at maximum speed in the high-speed benchtop microcentrifuge
A compact pellet will form of cellular debris; the plasmid should stay in solution.
5. Apply the supernatant from step 4 to the QIAprep column by decanting or pipetting
Try to not carry over any of the cellular debris matter as it can clog the column.
6. Centrifuge for 30–60s. Discard the flow-through.
The plasmid DNA will bind to the silica bed in the column. RNA, cellular proteins and metabolites are found in
the flow through.
7. Wash the QIAprep spin column by adding 0.5mL of Buffer PB and spinning for 30–60s. Discard the flowthrough. This step is necessary to remove trace nucleases.
8. Wash QIAprep spin column by adding 0.75mL of Buffer PE wash buffer and centrifuging for 30–60s
This step removes excess salts.
9. Discard the flow through, and centrifuge for an additional 1min to remove residual wash buffer
*Important: Residual wash buffer will not be completely removed unless the flow through is discarded before
this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
10a. Place the QIAprep column in a clean 1.5mL eppie. To elute DNA, add 50μL of Buffer EB (10mM Tris·Cl,
pH 8.5) or H2O to the center of each QIAprep column, incubate at RT for 5min, followed by a 1min spin.
EB is a low salt elution buffer. It is critical to incubate for at least the 5min as the purpose of this step is to
change the pH of the column allowing the release of the DNA into your eluent.
Quantifying Plasmid DNA:
1. Obtain OD260/OD280 readings of your mini-prepped DNA (in duplicate) using the Nanodrop™
spectrophotometer. The SOP is posted on the wall above the machine. You will be using EB Buffer as your
blank.
For each sample, you need to record the following information:
OD260
DNA
Sample ID
OD280
OD260/280
1.
1.
1.
2.
2.
2.
Mean
Mean
Mean
Questions (To be answered in your lab book):
1. Did you obtain the kit recommended concentration of DNA?
2. Is your DNA pure? If not, what is the likely contamination?
References
Birnboim H. C. and Doly J. (1979) A rapid alkaline extraction procedure for screening recombinant DNA.
Nucleic Acid Research 7: 1513-1523
Holmes D. S. and Quigley M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Analytical
Biochemistry 114: 193-197