Module 9: Extraction of Plasmid DNA and Restriction Mapping
1)
Introduction:
Biochemists study protein structure, function and activity. To study protein X, we need it in pure form rather than as a mixture of
many proteins. It is not always easy to purify a protein from its natural source. For example, to purify bovine protein X (from cow),
you might start by grinding up a piece of steak in a blender. Protein X may have low abundance and you would need many cows
worth of steak to get enough protein and this approach may not be practical. The advent of molecular biotechnology has simplified
protein purification. We can clone the gene that codes for protein X into a plasmid. We then insert this plasmid that now contains the
gene that codes for protein X into E. coli cells. The machinery inside the cells makes large amounts of protein X based on the
sequence of the gene. This is called overexpression.
The cornerstone of this technology is the plasmid. Plasmids are circular, small, extrachromosomal, autonomously replicating bacterial
DNA molecules that have become indispensable tools in recombinant DNA work, for gene cloning and expression. Interesting factoid:
The plasmid you will purify and study in this module is the same plasmid that was used to express the protein for the purification
module. This plasmid codes for B. stearothermophilus DHFR.
Restriction enzymes function like scissors. In this module, the instructor has done a restriction digest in which the plasmid was cut
with two restriction enzymes to release the “insert”. This is the part of the plasmid that contains the gene that codes for your protein of
interest. If you have a circle and you cut it once, it becomes linear. If
you cut
the circle twice, you get two linear pieces, the insert and the rest of the
plasmid
(sometimes called the vector). The size of the insert plus the size of the
vector
should be the size of the plasmid.
You will have access to a DNA ladder or DNA size standard. This will
in the same way as the protein ladder to determine the size of DNA
on the gel.
be used
pieces
2) Goal: You will receive a frozen pellet of E. coli cells containing a plasmid. Your goal is to determine the size of the plasmid and
the size of the insert within the plasmid. You will also need to determine whether or not the uncut, circular plasmid shows up
where you expect it to on the gel. If you know the size of the insert and vector without the insert, you can add them up to
determine the size of the whole plasmid. You need to do the following:
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Extract plasmid DNA from a frozen pellet of E.coli cells, (keyword: QIAGEN miniprep)
Quantitate the extracted DNA via spectrophotometry (Beer-Lambert law, using the relationship A260 = 1.0 for 50 ug/ml of
DNA) to figure out how much of your sample to load on the gel
Establish a simple restriction map of the plasmid by DNA gel electrophoresis
Determine the size of the DNA-insert and the vector without the insert via gel electrophoresis
A generic plasmid map outlining how the insert is cloned into the plasmid is supplied below.
3) Background Information:
• See the QIAGEN website for details and protocols. PDF of instruction booklet is also supplied. Important reminder: do
not let the SDS-NaOH lysis proceed for more than 5 minutes.
• To find the QIAGEN miniprep protocol, you can go to http://www.qiagen.com/products/catalog/sampletechnologies/dna-sample-technologies/plasmid-dna/qiaprep-spin-miniprep-kit#resources
• Once on this site (note you are under the resources tab), download the QIAprep Miniprep Handbook — May 2012.
• Find the basic description of the “Nanodrop” spectrophotometer online; the applicable Beer-Lambert law was discussed
in module 2.
• Because it takes a long time, the restriction digest will be prepared by the instructor ahead of time, and will be supplied
to you ready for the gel electrophoresis step. The restriction enzymes NdeI and BamHI were used for the plasmid
digestion. Check out the website for New England Biolabs (“www.NEB.com”), it has a lot of useful information
regarding restriction enzymes, restriction digests, etc.
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DNA-fragments in the agarose gels will be visualized by UV-induced fluorescence of ethidium bromide, using a UV
lightbox, WEAR GLOVES AT ALL TIMES WHEN PERFORMING THE ELECTROPHORESIS
Thought question: why do the small circular plasmid DNA molecules renature (i.e. reform doublestranded DNA) so much quicker
than the large genomic DNA pieces?
Principle of the “miniprep” procedure:
Based on their circular nature, plasmids can be easily isolated by a simple alkaline lysis procedure: bacterial cells containing the
plasmid are first resuspended in a neutral buffer, then a mixture of SDS-NaOH is added. SDS is a detergent that lyses the cell
membranes, whereas NaOH denatures the double-stranded DNA into single-stranded DNA.
Upon neutralization with NaAcetate most proteins in the lysate precipitate; the large chromosomal DNA molecules remain
denatured/single-stranded, while the small circular plasmid DNAs quickly renature to form double-strands. These double-strands are
then purified over a small silica-based minicolumn. See the Qiagen miniprep protocol for further details (ie. pages 7-11 for general
info and pages 19-22 for the specific protocol).
Important reminder: do not let the SDS-NaOH lysis proceed for more than 5 minutes!
The following was done by the instructor: Restriction mapping:
DNA (1-2 ug) are digested with a restriction enzyme, and the sizes of the resulting DNA fragments are analyzed by agarose gel
electrophoresis. In order to save time, this sample will be provided to you already prepared.
Here is a general outline of how one would setup such a digest with about 1ug of DNA in a final volume of 20 µL. Supplied are a 10 x
concentrated restriction enzyme digestion buffer and the restriction enzyme. Assuming a DNA concentration of 300ng/µL, as an
example, a 20µL digestion would be setup as follows:
Addition
DNA
10x buffer
Water
enzyme
Volume (µL)
3
2
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4) Math moment:
1) When A260 = 1, the concentration of DNA is 50 ug/ml. What would the A260 of a 2.5mg/ml DNA stock solution be? Note:
A260 = absorbance at 260 nm. Note, the absorbance is proportional to concentration.
2) How do you prepare 5 ml of a DNA solution with a concentration of 30 ug/ml, using a DNA stock solution supplied at 1.25
mg/ml? Please, clearly and thoroughly explain the steps you would take to accomplish this.
3) You have a DNA solution at a 75 ug/ml concentration. How do you prepare a dilution that would have an A260 = 0.12?
5) Protocol outline:
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DNA extraction: see Qiagen manual pages 19 to 22
DNA quantitation by A260: performed by the instructor using the Nanodrop spectrophotometer; blank the instrument against
the Qiagen miniprep elution buffer; record A260, A280 and A320; calculate concentration of DNA prep using the Beer-Lambert
law, using the relationship A260 = 1.0 for a 50 ug/ml of DNA) and
Gel electrophoresis: small 1% agarose gels in 1 X Tris-Acetate buffer (“running buffer”) will be supplied. The buffer and the
gels contain ethidium bromide for DNA staining, WEAR GLOVES AT ALL TIMES WHEN PERFORMING THE
ELECTROPHORESIS. To prepare your samples for electrophoresis, mix 4 µL of the blue loading dye (“blue juice”) with about
2
2 µL of your plasmid DNA preparation (discuss with instructor), and load the entire sample into one gel slot, along with 10 µL of
the DNA size marker and 10 µl of the instructor-prepared plasmid digest. Connect the leads to the power supply and run the gel at
about 80 V for 45 minutes to one hour. Remove the gel from the box, place on the UV lightbox, turn UV light on and take a
picture using your cell phone camera.
6)
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Relevant Boyer Book Chapters:
#6 (electrophoresis)
#7 (spectrophotometry)
#9 (plasmid DNA isolation)
#10 (restriction mapping)
7) Supplies provided:
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Qiagen miniprep materials (tubes, buffers, mini columns, microcentrifuge, )
Frozen pellet of E. coli cells for DNA extraction
Micropipets (P1000, P200, P20) and tips
Nanodrop spectrophotometer (instructor will perform the measurements)
The restriction digest prepared by the instructor for the gel electrophoresis step. The restriction enzymes NdeI and
BamHI were used for the plasmid digestion.
Agarose gels, small electrophoresis boxes, electrical leads, power supplies, DNA size marker (see
https://www.neb.com/products/n3232-1-kb-dna-ladder), loading dye (“blue juice”)
Generic plasmid map
8) Vocabulary:
Plasmid, restriction enzyme, open reading frame, promoter
9) Plasmid map: See next page.
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Figure 1. Generic plasmid map. Note the open reading frame labeled DHFR. This is the “insert” in this plasmid. Note how the insert
is flanked by two restrictions sites (NdeI and BamHI). Note, there is only one NdeI and one BamHI site in this plasmid. This means
that if you cut with NdeI, you get the same size DNA piece as the plasmid but it is linear instead of circular. If you cut with BamHI,
you also get a single linear piece. If you cut with both, you get two pieces (DHFR piece and the rest of the plasmid DNA). The
restriction enzymes NdeI and BamHI were used for the plasmid digestion in the sample provided for you today.
10) Datasheet to hand in:
Each group is to hand in a datasheet (one week after the experiment was performed) with the following information:
• A260 reading of your DNA prep, calculation of DNA concentration
• Semilog plot of the DNA standards used on the gel: plot log [DNA sizes in bp] vs [migration in mm]; then estimate the
size of the insert released from the plasmid (instructor-supplied sample) and the vector without the insert. Finally, from
that information determine the size of the plasmid before it was cut. Comment on whether the circular (uncut) plasmid
you purified runs where you would expect it to on the gel.
11) Safety
WEAR GLOVES AT ALL TIMES WHEN PERFORMING THE ELECTROPHORESIS. DO NOT TOUCH THE GEL WITHOUT
GLOVES ON. DO NOT TOUCH THE ELECTROPHORESIS BOX OR POWERSOURCE WHEN THE POWER IS ON.
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