Download Extraction of Plasmid DNA, Restriction Digest, and DNA Gel

Module 9: Extraction of Plasmid DNA, Restriction Digest, and DNA Gel Electrophoresis
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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
be used in the same way as the protein ladder to determine the size of
DNA pieces on the gel.
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.
A generic plasmid map outlining how the insert is cloned into the plasmid is supplied below.
Hint: You will need to carry out protocols to
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extract plasmid DNA from a frozen pellet of E.coli cells, (keyword: QIAGEN miniprep)
you might 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
determine the size of the DNA-insert and the vector without the insert via gel electrophoresis
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 doublestrands 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!
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. Relevant Boyer Book Chapters:
• #6 (electrophoresis)
• #7 (spectrophotometry)
• #9 (plasmid DNA isolation)
• #10 (restriction mapping)
6. 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
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7. Plasmid map:
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.
8. 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.
9. 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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