BIOL 220 Protocol 3 Isolating Plasmid DNA

Isolating plasmid DNA from bacterial (E. coli) culture

Background
Plasmids are double stranded pieces of DNA that can replicate within bacterial cells independently of the
cells chromosome. Plasmids occur naturally in many bacteria, but can also be modified in the lab to aid in
cloning foreign DNA of interest. The plasmid can be replicated in special strains of bacteria such as
Escherichia coli and then purified for downstream applications such as sequencing. Plasmids are three orders of
magnitude smaller (2-4 kb) than the typical bacterial chromosome, such as E. coli (4.6 Mb). Our spider cDNAs
were ligated into the pZeR0-2 (Invitrogen) vector to create a recombinant plasmid. Based on the size of the
cDNA insert (see Protocol I Quick Size Screen) we have chosen clones from the cDNA library for which we
wish to isolate the plasmid DNA.
A popular method for isolation of plasmid DNA is alkaline lysis (Birnboim and Doly, 1979; Birnboim,
1983). The goal is to remove E. coli cellular debris, chromosomal DNA, proteins, and cellular RNA, leaving a
purified plasmid with little contamination. This method exploits the fact that after cell lysis chromosomal DNA
tends to break up into linear fragments, while the small plasmid remains a covalently closed circular piece of
DNA. Essentially, this means that the plasmid remains supercoiled with no breaks in the sugar-phosphate
backbone. The first step in the protocol is to lyse the cells under alkaline conditions (pH 11) in the presence of a
detergent (SDS). High pH causes double stranded DNA to denature. The lysate is then rapidly neutralized with
a high salt buffer. Linear fragments of chromosomal DNA will base pair in an intrastrand manner, forming
insoluble aggregates. However, the circular plasmid molecules will anneal with their appropriate complement
reforming the original double stranded DNA. These molecules stay in solution. Furthermore, the proteins and
detergent precipitate and entrap very large chromosomal DNA and cellular RNA fragments. The soluble
plasmid DNA is then physically separated from the precipitated materials (lysate), usually by centrifugation.
The plasmid DNA can be further concentrated by binding to silica and/or by ethanol precipitation. The
protocol we will use takes advantage of both methods. DNA has a negative charge and is usually soluble in
water, which is also polar. However, ethanol is not as polar as water. In a high enough concentration of
ethanol, the negatively charged phosphate backbone will bind with positive ions and precipitate out of solution.
Positive ions, usually Na+, NH4+, K+, or Li+, are supplied by an appropriate salt solution. The precipitated DNA
will form a solid pellet upon centrifugation. Alternately, the DNA solution can be passed through a silica
membrane to aid purification and concentration. DNA will bind silica in the presence of chaotropic salts. A
chaotrope such as guanidium hydrochloride denatures biomolecules by removing water. This allows positively
charged ions (e.g. K+) to form a bridge between the negatively charged DNA and the negatively charged silica.
Once the DNA is bound to the silica, contaminants that do not bind to the silica membrane can be removed by
washing in a high salt, ethanol solution. The DNA can then be released from the silica membrane by applying a
low salt solution, such as pure water.
Literature Cited
Birnboim, H.C., Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant
plasmid DNA. Nucleic Acids Research 24: 1513-15-23.
Birnboim, J. 1983. A rapid alkaline extraction method for the isolation of plasmid DNA.
Methods of Enzymology. 100: 243-255.

BIOL 220 Protocol 3 Isolating Plasmid DNA

Protocol - Plasmid isolation by alkaline lysis and silica binding
This protocol uses the QIAprep Miniprep kit (Qiagen). Detailed information on this kit can be found at
http://www.qiagen.com.
Materials
Buffer P1 = Cell Resuspension Solution (50mM Tris-HCl pH 7.5, 10mM EDTA, 100ug/ml RNase A)
Buffer P2 = Cell Lysis Solution (0.2M NaOH and 1% SDS, pH 11)
Buffer N3 = Neutralization Solution (4.09M guanidine hydrochloride, 0.759M potassium acetate, 2.12M glacial
acetic acid, pH 4.2)
Buffer PB = proprietary buffer
Buffer PE = Column Wash Solution (60mM potassium acetate, 8.3mM Tris-HCl pH7.5, 40uM EDTA pH 8.0,
60% ethanol)
Buffer EB = Elution Buffer (10 mM Tris)
Spin Columns
Collection Tubes (2 ml)
Sterile 1.5 ml centrifuge tubes
Pipettes and tips
Centrifuge
Production of cleared lysate
1. Label an appropriate number of 1.5 ml centrifuge tubes with the clone name found on the glass culture
tube (e.g. PM25). (Note be sure to write in your lab notebook the names of the clones you are
working with).
2. Transfer 1.4 ml of E. coli culture from the glass tube to the 1.5 ml tube. (Set your pipetter to 700 ul and
pipette twice; or pour).
3. Centrifuge your samples for 3 minutes at ~7,000 RCF. Be sure to BALANCE the centrifuge.
4. Carefully pour off the supernatant (the liquid) into an appropriate container (small beakers on your
bench labeled bacterial waste). Watch the pellet at the bottom of the tube to make sure it doesnt
become dislodged. Blot excess liquid with a clean paper towel or kimwipe.
5. Repeat steps 2-4 to pellet the entire bacterial culture in a single 1.5 ml tube. (Do NOT mix samples!)
6. Add 250 ul of Buffer P1 to your pelleted cells.
7. Completely resuspend the cells by vortexing for at least 1 minute. It is essential to thoroughly
resuspend the cells.
8. Add 250 ul of Buffer P2 (lysis solution) to resuspended cells.
9. Mix by inverting the tube 4-6 times. Do NOT vortex!!!
The cell suspension should turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored
suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still visible,
continue mixing the solution until a homogeneously colored suspension is achieved.

10. Continue inverting tube until the cell suspension clears and color is homogenously blue (2-5 minutes).
Do NOT allow lysis to proceed for longer than 5 minutes.
11. Add 350 ul of Buffer N3 (Neutralization Solution).
12. Immediately mix by inverting the tube 4-6 times.
The suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous
colorless suspension indicates that the SDS has been effectively precipitated.

13. Centrifuge the tube at ~17,900 RCF for 10 minutes.

A compact white pellet will form that includes cellular debris, E. coli genomic DNA, proteins, fats, and other
unwanted material. The liquid contains the plasmid DNA.
You may wish to clean your glass culture tubes during centrifugation. (See Clean-up below).

BIOL 220 Protocol 3 Isolating Plasmid DNA

Binding plasmid DNA to silica
14. While your samples centrifuge Obtain a Spin Column inside a 2ml collection tube for every sample
you are processing. Label the Spin Columns and Collection Tubes with the appropriate clone name.
15. Transfer the supernatant from step 13 into the Spin Column by careful pouring. Avoid transferring or
disturbing any of the white lysate (the pellet).
NOTE: If the white precipitate is accidently transferred to the Spin Column pour the contents back
into the 1.5ml tube and centrifuge again. Also tell Dr. Ayoub.

16. Centrifuge the supernatant in the Spin Column plus collection tube for 1 minute at 17,900 RCF.
17. Remove the Spin Column from the collection tube and discard the flow through into bacterial
waste container. Reinsert the Spin Column into the collection tube.
Washing plasmid DNA
18. Add 500 ul of Buffer PB to the Spin Column.
19. Centrifuge at 17,900 RCF for 1 minute.
20. Remove the Spin Column from the collection tube and discard the flow through into the bacterial
waste container.
21. Reinsert the Spin Column into the collection tube.
22. Add 750 ul of Buffer PE (Wash Solution) to the Spin Column.
23. Repeat steps 19&20.
24. Centrifuge at maximum speed for 1 minute to completely dry the silica membrane.
Elution
25. Transfer Spin Column to a new, sterile LABELLED 1.5 ml tube. (It is OK to discard the Collection
Tube at this point).
NOTE: Be careful to NOT transfer any of the wash solution with the Spin Column. If the Spin
Column is not completely dry, centrifuge an additional 1 minute. Then transfer Spin Column to
another 1.5 ml tube.

26. Add 50 ul of Buffer EB to the Spin Column. Carefully pipette the water directly onto the silica
membrane. Do NOT touch the membrane or the walls of the column.
27. Centrifuge at 17,900 RCF for 1 minute.
28. Check that all of your tubes have ~50 ul in the bottom of the tube. Discard Spin Column.
29. Label the side of the 1.5ml tube with the clone name, your initials, and the date (e.g. PM25 NAA
24Sep13).
30. Proceed to Restriction Digest or sample storage.
31. Give your sample(s) to Dr. Ayoub for storage at -20C.
Clean-up
Prior to proceeding with any further lab work it is important to complete the following clean-up steps.
1. Clean and dispose glass culture tube:
a. Squirt a small amount of 10% bleach into each of your glass culture tubes. Let sit at least five
minutes (Note: a good time to squirt with bleach is while your samples are spinning in step 13).
b. Rinse glass tubes with tap water.
c. Dispose of glass tubes in glass waste container (see Dr. Ayoub for location).
2. Pour contents of bacterial waste container into a single provided container. Rinse with tap water.
3. Place all reagents back where you found them.
4. Wipe your bench with 10% bleach and paper towels or with a Clorox wipe.
5. Throw away your gloves and get fresh gloves.
These steps are crucial to prevent the spread of genetically modified E. coli bacteria.