Biochem 121.

1 Plasmid DNA Extraction and Restriction Enzyme Digestion Page 1 of 8

Biochem 121.1 Biochemistry of the Gene Laboratory
Plasmid DNA Extraction and Restriction Enzyme Digestion
2010-00269, 2010-29246, 2010-48759
Group 4, Ms. Joanne Del Rosario
Submitted February 17, 2014
Abstract
Plasmids are extra-chromosomal, double-stranded, circular DNA molecules that carry genes that
code for a wide array of traits that offer organisms genetic advantages, such as antibiotic resistance.
Plasmids are most useful in recombinant DNA technology and in analyses in molecular biology and
biochemistry, such as restriction enzyme digestion. Restriction enzymes are enzymes that cleave the
DNA sequence at specific nucleotide sequences, making it possible to extract only specific parts of the
DNA which are of interest. In this experiment, plasmid DNA was extracted from E. coli. in a procedure that
involved alkaline lysis, rapid denaturation and renaturation of chromosomal DNA and its subsequent
separation from plasmid DNA. The extracted DNA was then subjected to restriction enzyme digestion by
EcoR1. Four samples of digested DNA incubated at 15, 30, 45 and 60 minutes respectively, were made
to undergo agarose gel electrophoresis together with the undigested extracted DNA sample. AGE results
show that all four digests showed no difference in size and molecular weight, as all of them showed a
distinct band of size 2 kbp and molecular weight of 40 ng. The undigested plasmid DNA, however, had a
molecular weight of 120 ng. This suggests that the reaction time allotted in the procedure may not have
been enough for complete digestion to occur.
Keywords: plasmid DNA extraction, restriction enzyme digestion, alkaline lysis, restriction site, agarose
gel electrophoresis
I. Introduction
Virtually all bacterial species contain
plasmidsdouble-stranded, typically circular DNA
molecules distinct from the main chromosome in a
cell. Plasmids account for only a small fraction of
the bacterial genome corresponding roughly to a
range between 1 and 200 kb. A plasmid of more
than 50 kb may be characterized as large, and
one of less than 10 kb may be called small.
Plasmids are capable of being stably inherited
without being linked to the chromosome and can
be transferred horizontally between cells between
same species or even between different species.
(Rohde & Henze, 2011, Rhodes, et al., 2004,
Plasmid Isolation, 2014)
One of the most important aspects of
bacterial plasmids is their carriage and spread of
antibiotic resistance genes that ultimately have an
impact on the treatment of diseases of animals
and humans. They can also carry genes that code
for a wide range of metabolic activities, enabling
their host bacteria to degrade pollutant
compounds, and produce antibacterial proteins.
They can also harbor genes for virulence that help
to increase pathogenicity of bacteria causing
diseases such as plague, dysentery, anthrax and
tetanus. Because of their ability to carry genes
that provide the cell with genetic advantages,
plasmids are used in recombinant DNA
experiments to clone, transfer and manipulate
genes from other organisms and make large
quantities of their DNA. (Rhodes, et al., 2004,
Plasmids, 2013)
Plasmid DNA extraction and purification is
a cornerstone of many molecular biology and
biochemistry labs due to its extensive use prior to
a variety of applications such as transfection,
bacterial transformation, polymerase chain
reaction, sequencing, in vitro transcription and
restriction endonuclease digestion. Over the
years, many methods for plasmid DNA extraction
have been made; however, most of these
methods have compromisessome can give a
Biochem 121.1 Plasmid DNA Extraction and Restriction Enzyme Digestion Page 2 of 8
low yield, may be time-consuming and produce
impure plasmid extracts. In 1979, scientists
Birnboim and Doly proposed a method of
extracting plasmid DNA involving selective
alkaline denaturation of chromosomal DNA while
circular DNA remains double-stranded. To this
day, alkaline lysis is the preferred method for
plasmid DNA extraction from bacteria. (Plasmid
DNA Purification, n.d., Birnboim & Doly, 1979)
Plasmid DNA extracted using Birnboim
and Doly is pure enough to be subjected to
restriction enzyme digestion. Restriction enzymes,
also called restriction endonucleases, are
enzymes that cut the sugar-phosphate backbone
of DNA molecules. In order to be able to
sequence DNA, it is first necessary to cut it into
small fragments. Many DNA-digesting enzymes
can accomplish this; however, what is needed is a
way to cleave the DNA at specific sites to produce
a small set of homogenous fragments. This is why
restriction enzymes are of particular importance;
they recognize and cut DNA only at a particular
sequence of nucleotides. Most recognition
sequences are palindromesthey read the same
forward (5 to 3) and backward (3 to 5). Also, the
cuts made by restriction enzymes can generate
either sticky ends (with 5 and 3 overhangs)
which were cut asymmetrically, or blunt ends
which were cut at precisely opposite sites.
(Bowen, 1999, Restriction Enzymes, 2013)
In this experiment, plasmid DNA was
extracted from bacteria in a procedure that used
alkaline lysis, and the extracted plasmid DNA
underwent restriction enzyme digestion before
being subjected to agarose gel electrophoresis.
II. Experimental
A. Plasmid DNA Extraction
Three solutions were made with their
respective contents; Solution 1: 50 mM glucose,
25 mM Tris-Cl at pH 7.4 and 10 mM EDTA at pH
8.0; Solution 2: 1 M NaOH and 10% SDS;
Solution 3: 6 mL of 5 M potassium acetate, 1.5
mL glacial acetic acid and 2.5 mL sterile distilled
water.
A single bacterial colony was transferred
into 2 mL of LB-ampicillin medium and incubated
overnight at 37
o
C with vigorous shaking. From the
overnight culture, 1.5 mL was pipetted into a
microcentrifuge tube. The tube was then
centrifuged at 12000xg for 1 minute at 4C. The
medium was completely removed by aspiration.
The bacterial pellet was resuspended in
400 L of ice-cold Solution 1. The solution was
pipetted up and down with a pipette tip to
completely resuspend cells. Next, 400 L of
freshly prepared Solution II was added. The tube
was closed tightly, and the contents were mized
by inverting the tube rapidly but gently five times.
The tube was stored on ice for 3 minutes. Three
hundred microliters of ice-cold Solution III was
then added. The tube was closed and vortexed
gently in an inverted position for 10 seconds to
disperse the solution through the viscous bacterial
lysate. The tube was stored on ice for 5 minute
without shaking. The tube was then centrifuged at
12000xg for 5 minutes at room temperature. The
supernatant was recovered and the pellet
discarded.
The supernatant was divided into 500 L
aliquots in 1.5 mL microcentrifuge tubes. Two
volumes of absolute ethanol were added to it at
room temperature. The solution was mixed by
vortexing and let stand for 5 minutes at room
temperature. The tube was centrifuged at
12000xg for 5 minutes at 4C. The supernatant
was removed by gentle aspiration and discarded.
To the precipitate, 600 L of 70% ethanol
was added. The tube was again centrifuged at
12000xg for 5 minutes at 4C and the supernatant
was removed by gentle aspiration and discarded.
Another round of 600 L of 70% ethanol was
added to the precipitate. The pellet was air-dried
and was resuspended in 100 L TE buffer, ready
for storage.
B. Restriction Enzyme Digestion
A master mix for 10 reactions was
prepared so that each 0.2mL PCR tube contained
2.5 L 10X RE buffer, 18.75 L sddH
2
O, 2.5 L of
the extracted plasmid from the previous
experiment, and 1.25 L restriction enzyme,
EcoR1 (10 U/L).
Biochem 121.1 Plasmid DNA Extraction and Restriction Enzyme Digestion Page 3 of 8
The tubes were incubated at 37C for 1
hour. For every 15 minutes, an aliquot (5 L of the
RE reaction) was obtained and loaded to 1%
agarose gel together with 1 L of 6X gel loading
buffer. AGE was run at 100 volts for 40 minutes.
Undigested sample was also included to compare
uncut, partially digested, and completely digested
plasmid DNAs.
III. Results and Discussion
Plasmid DNA Extraction
Plasmids are small, extra- chromosomal
genetic elements that are found in almost all
bacterial cells and also in some eukaryotic
organisms (Sambrook and Russell, 2001). These
are double stranded and circular DNA molecules
that range from 1 kb to greater than 200 kb in size
(Lipps, 2008). Plasmids are considered to be
among the most important contributors in the
evolution of prokaryotes as they can serve as
vehicles that carry artificially inserted DNA and
they provide an essential role in gene
manipulation.
Plasmids are also considered to be
originators of several drug resistance
mechanisms. Some strains of bacteria can be
resistant to antibiotics either by acquiring several
independent plasmids or through acquisition of a
single plasmid with many sources of resistance on
it. In this experiment, E. coli was used to
assimilate the pGEM-1 plasmid which carries the
ampicillin resistance gene (pGEM-1, 2011). To
better analyze and observe plasmid DNA, a
molecular biology technique known as plasmid
DNA isolation is employed. In general, this
procedure involves four steps: (1) immediate lysis
of the bacterial cells, (2) separation of the plasmid
from the chromosomal DNA, (3) removal of the
cellular components that may alter subsequent
test results and lastly, (4) removal of the
detergents and salts added in the process
(Kieleczawa, 2006).
An ampicillin resistant E. coli was used as
the specific host of interest and such phenotype
was made by adding the antibiotic during the
overnight culture preparation. This is to carefully
select against the cells that do not produce the -
lactamase protein and thus, the ones which are
not resistant to the effects of Ampicillin. The gene
for antibiotic resistance produces the -lactamase
that breaks down any chemical with a beta-lactam
ring. Through this, the antibiotic around the E. coli
will become degraded and the bacteria will be
able to survive. The extraction procedure utilized
in this experiment is called the alkaline lysis
method. The alkaline lysis preparation is
considered to be the most commonly used
method for isolating small amounts of plasmid
DNA. Procedures such as this are generally
based on the fact that plasmids usually occur in
the covalently closed circular configuration within
the host cells.
Figure 1. A schematic representation showing the mechanism
of resistance of bacteria to penicillin, an ampicillin- like
antibiotic. Retrieved from: http://www.wiley.com/college/
pratt/0471393878/instructor/activities/bacterial_drug_resistanc
e/index.htmlssss
Three solutions having their own specific
modes of action were prepared and used to
facilitate the complete lysis of the bacterial cells.
Solution 1, comprising of glucose, Tris-Cl and
Ethylenediaminetetraacetic acid (EDTA), is
considered to be the resuspension buffer and has
a mechanism that is mainly directed towards the
cell wall. Glucose functions to maintain the
necessary osmotic pressure for the rest of the
reactions to proceed. It usually makes the solution
isotonic. However, for most bacteria including E.
coli DH5a, isotonicity is not required since they
contain cell wall that can withstand a wide range
of solution concentration (Boffey, 1983). With the
use of Tris-Cl and EDTA, cell lysis is further
enhanced as they disrupt the outer envelope and
expose the peptidoglycan. Tris is known as an
ideal buffering agent as it buffer the cell at pH 8
while EDTA chelates with divalent cations (Ca
2+
,
Biochem 121.1 Plasmid DNA Extraction and Restriction Enzyme Digestion Page 4 of 8
Mg
2+
, Mn
2+
) which are released upon bacterial
lysis. The action of EDTA results in inactivation of
many enzymes which may possibly harm the
plasmid DNA if not removed (Birnboim and Doly,
1979).
The Alkaline lysis solution 2 was a
freshly-prepared combination of SDS and NaOH.
Sodium dodecyl sulfate is a weak, anionic
detergent that dissolves the lipid components of
the cell membrane and cellular proteins.
Denaturing action of SDS also releases proteins
from DNA, leaving the DNA free from
contaminating proteins. Conversely, sodium
hydroxide denatures both the chromosomal and
plasmid DNA into single strand by weakening the
hydrogen bonds that hold the two strands
together (Birnboim and Doly, 1979). It is of great
importance that this solution is freshly prepared to
take into account the presence of NaOH and the
tendency of water to react with carbon dioxide. If
not freshly prepared, it is of great possibility that
NaOH can also react with carbon dioxide. Carbon
dioxides reaction with alkalis can lead to the
production bicarbonates whereas its response to
water is to produce carbonic acid (Pauling, 1988).
These two reactions are illustrated below:
CO
2
+ H
2
O H
2
CO
3

CO
2
+ NaOH NaHCO
3

The last solution, solution 3, contains
potassium acetate and acetic acid. The addition of
acetic acid neutralizes the high pH due to NaOH
so that DNA strands can rapidly renature.
Potassium ions interact with the SDS to make this
detergent insoluble from the solution. The SDS
then easily precipitates and is separated through
centrifugation. Furthermore, the insoluble SDS
traps the larger genomic DNA to remove it from
the supernatant. This enables the plasmid DNA to
remain in solution (Birnboim and Doly, 1979).
Aside from the fact that biological activity
is maintained at approximately 4
o
C, it is essential
for solutions 1 and 3 to be stored in an ice-cold
environment so that each of them can serve its
purpose well. Note that storing solution 2 in such
environment is not that compulsory since it is
expected that it is freshly prepared. Maintaining
this temperature for Solution 1 inhibits the
nucleases that may cleave the plasmid DNA of
interest. Conversely, for Solution 3, the
precipitation of the chromosomal DNA and the
SDS-protein complexes is facilitated at this
temperature. Also, since DNA is very sensitive to
mechanical stress, shearing forces caused by
techniques such as vortexing and fast pipetting
are avoided as soon as cell lysis occurs. All
mixing steps during and after cell lysis was
carefully performed by inverting the tubes several
times. Vigorous vortexing is avoided as it can
damage or shear the extracted DNA (Clewell,
1972).
A primary alcohol, such as ethanol is the
most commonly used component in the
precipitation of DNA. Due to the structural
differences between ethanol and water, ethanol
has much lower dielectric constant than water
does (Maniatas, Fritsch, Sambrook, 1982). Water
is considered to have a high dielectric insulator,
which means that the electrostatic force existing
between two ions of opposite charge is very low in
comparison with that in ethanol. Adding ethanol
lowers the dielectric constant in the solution so
water no longer insulates individual ions. Because
of this, the Coulomb force of attraction increases
between the cations and the negatively charged
nucleic acid backbone. Once their interaction
begins, nucleic acids are neutralized such that
they no longer dissolve in water and hence are
precipitated out of solution (Pikur and Rupprecht
1995).
Afterwards, the isolated DNA was
suspended in a Tris-EDTA buffer for long-term
storage. The EDTA in TE buffer chelates
Mg
2+
and other divalent metal ions responsible for
DNA and RNA degradation, thus suppressing
these processes (Birnboim and Doly, 1979).
Restriction Enzyme Digestion
Restriction endonucleases or enzymes are
naturally occurring enzymes that cut double
stranded DNA at specific sites called recognition
sequence which are usually 4-8 nucleotides long
and are palindromes, meaning that the sequence
reads the same when read from 5 to 3' at each
strand. Restriction endonucleases cleave DNA by
Biochem 121.1 Plasmid DNA Extraction and Restriction Enzyme Digestion Page 5 of 8
catalyzing the hydrolysis of the sugar-phosphate
backbone (Berg, Tymoczko, & Stryer, 2002).
Restriction enzymes are isolated from
different bacterial species wherein they play a role
in the defense mechanism of the bacteria against
invading foreign DNA such as viruses. Restriction
endonucleases, together with the enzyme
methylase, comprise the host-controlled
restriction modification systems of certain
bacterial species including Escherichia coli.
When a bacteriophage inserts its DNA
into the host cell, the endonuclease synthesized
by that cell would cleave the foreign DNA thereby
restricting the infecting virus. The host DNA
however contains the same recognition sequence,
so the cell produces a corresponding methylase
which would add methyl groups to cytosine or
adenine bases to protect the host DNA from the
action of its own endonuclease (Kumar & Garg,
2005).
There are of four types of restriction
ennzymes namely, Type I, Type II, Type III and
Type IV which differ in their recognition sequence,
subunit composition, cleavage position, and
cofactor requirements. Type I and Type III are
large, multi-subunit enzymes that cut DNA at
random, far from their recognition sequences;
Type I cuts DNA locations as far as 1000 or more
base-pairs from the recognition site while Type III
cuts at approximately 25 base-pairs from the site.
Both types carry restriction and methylase
activities and are ATP dependent.
Type II restriction enzymes cut DNA
within or with a short distance from the recognized
sequence and are the predominantly used
restriction enzyme in DNA analysis and cloning
studies. They are smaller and simpler, with
subunits of 200 to 350 amino acids. Type II
enzymes do not require ATP. Type IV enzymes
recognize modified or methylated DNA. Type I, II,
and III all requires a cofactor, usually Mg
2+
. (New
England Biolabs, 2014)
Upon RE digestion, DNA may generate
blunt ends, 5 or 3 sticky ends or overhangs
depending on the position of the cutting site within
the recognition sequence (Kumar & Garg, 2005).
Cleaved DNA sequences with sticky ends will
easily anneal through ligation than those with
blunt ends. Hence, restriction enzymes which
produce sticky ends or overhangs are of great use
in the recombinant DNA technology.
In this experiment, a plasmid DNA
isolated from E. coli was subjected to restriction
enzyme digestion. The restriction enzyme used is
EcoR1, a Type II enzyme which produces 5
overhangs. EcoR1, isolated from Escherichia coli
RY13, has a recognition sequence and restriction
site shown below:



If the plasmid has only one GAATTC
sequence, EcoRI would cut the plasmid once,
converting it to a linear piece of DNA. When
visualized using agarose gel electrophoresis, a
single band should be observed having a
molecular weight same as that of the extracted
plasmid DNA. Presented in the results in Figure 1,
all four RE digests showed a distinct band with
size of 2 kbp and molecular weight of 40 ng. This
result however is inconsistent with the molecular
weight of the plasmid DNA which is 120 ng.

Figure 1.Plasmid DNA extract and RE digest resolved under
1.0 % agarose gel. Lane 1 represents the 2-log DNA ladder,
Lanes 2 and 3, extracted plasmid DNA and Lanes 4, 5, 6 and
7 the restriction enzyme digest at 15, 30, 45 and 60 minutes,
respectively.
Biochem 121.1 Plasmid DNA Extraction and Restriction Enzyme Digestion Page 6 of 8
Each restriction enzyme has optimal
reaction conditions; factors including pH, reaction
temperature and time, and enzyme concentration
affect the results of DNA digestion. For EcoR1
and most other restriction enzymes, the optimal
pH of the RE buffer is 7.5 and incubation
temperature is 37 C; these conditions generally
reflects the growth requirement of the bacterial
strain from which the enzyme is derived.
Incubation time was set to one hour, and
to monitor the restriction enzyme activity, aliquots
of the reaction mixture were obtained every fifteen
minutes and then loaded on the agarose gel. The
results then suggest that the reaction time might
not have been enough for complete digestion.
Under optimal conditions the difference
there is a high difference between the cleavage
rates at the recognition or cognate site and the
next best site (with a single base substitution).
The rate difference of EcoRI at its cognate site
(5-GAATTC-3) and next best site (5-TAATTC-
3) is of the order of 10
5
(Promega, 2014).
However, when restriction enzyme digestion is
done under non-standard or non-optimal
conditions, the specificity of the endonuclease
decreases and would cleave the DNA at sites
different from the normal recognition sequence.
This non-specific cutting is called "star activity", a
property which is exhibited by EcoRI. Factors that
cause star activity include extremely high
concentration of the restriction enzyme, prolonged
reaction time, non-optimal pH, and presence of
organic solvents (New England Biolabs, 2014).
Restriction endonucleases as well as
many other enzymes that act on phosphate-
containing substrates require Mg2+ or some other
similar divalent cation for activity (source: journal).
However, use of other divalent cation such as
Mn
2+
, Co
2+
, and Fe
2+
may also cause relaxation of
specificity, and consequently result to star activity.
This is because other divalent cations may not fit
correctly into the active site of the restriction
enzyme, interfering with proper recognition (New
England Biolabs, 2014).
Appearance of extra bands on the
agarose gel can be attributed to star activity, as
well as to partial digestion, wherein longer
reaction time is recommended, and also to the
binding of enzyme to substrate DNA, which could
be resolved by lowering the enzyme concentration
or by adding SDS to the loading buffer to separate
the enzyme and substrate.
Other problems encountered in RE
digestion include incomplete restriction enzyme
digestion or no digestion at all, which could be
due to insufficient enzyme concentration, short
reaction time, or may be due to methylation; the
plasmid DNA isolated from E. coli may be blocked
by Dam and Dcm methylation, hence blocking the
cleavage by endonucleases. Another is having
diffused DNA bands or smears which could be
attributed to contamination or to enzyme-
substrate binding as well (Fermentas Life
Sciences, 2014).
IV. Conclusions and Recommendations
The method used for plasmid DNA
extraction involved the addition of three solutions
with distinct roles: the first solution is a buffer that
resuspends the DNA in optimum conditions to
prevent degradation; the second solution
separates contaminating lipids and proteins from
the DNA and denatures the DNA; and the third
solution rapidly renatures the DNA and separates
the chromosomal DNA from the plasmid DNA.
The extracted plasmid DNA was then made to
undergo restriction enzyme digestion before being
subjected to agarose gel electrophoresis. The
results suggest that an hour of reaction time was
not able to completely digest plasmid DNA, and
that 15-min. incubation intervals would not make a
significant difference in the rate of digestion. This
also suggests that standard and optimum
conditions may not have been achieved during
restriction enzyme digestion.
V. References
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15, 2014 from http://www.wiley.
com/college/pratt/0471393878/instructor/
activities/bacterial_drug_resistance/index.
html
Birnboim, H.C. and Doly, J. 1979. A rapid alkaline
procedure for screening recombinant
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plasmid DNA. Nucleic Acids Res. 7:
15131523.
Boffey, S. (1983). Techniques in Molecular
Biology. In J. Walker, & W. Gaastra.
Bristol: Leaper and Gard Ltd.
Bowen, R. (1999). Biology and Activity of
Restriction Endonucleases. Retrieved
February 16, 2014 from http://www.viv
o.colostate.edu/hbooks/genetics/biotech/e
nzymes/renzymes.html
Clewell. D.B. 1972. Nature of ColE1 plasmid
replication in Escherichia coli in the
presence of chloramphenicol. J. Bacteriol.
110, 667-676.
Ethanol precipitation of DNA with salts - Theory.
Retrieved February 16, 2014, from
http://www.nhm.ac.uk/resources-
rx/files/ethanol-precipitation-of-dna-with-
salts---theory_aug12-118483.pdf
Kieleczawa, J. (2006). DNA sequencing II:
Optimizing Preparation and Cleanup,
Volume 2. Jones & Bartlett Learning.
Kumar, A., & Garg, N. (2005). Genetic Enginee-
ring. Nova Science Publishers, Inc.
Lipps, G. (2008). Plasmids: Current Research and
Future Trends. Horizon Scientific Press.
Maniatis, T, E F Fritsch, and J Sambrook.
Molecular Cloning. A Laboratory Manual.
New York: Cold Spring Harbor
Laboratory, 1982.
Pauling, L. (1988). General Chemistry. Courier
Dover Publications.
pGEM-1. (2011). Retrieved February 16, 2014,
from LabLife: http://www.lablife.org/
p?a=vdb_view&id=g2.3ZIPOlP1R3J8RfrF
DDsGU4r9YcA-
Pikur, Jure, and Allan Rupprecht. "Aggregated
DNA in ethanol solution." FEBS Letters
375, no. 3 (Nov 1995): 174-8.
Plasmid DNA Purification (n.d.). Retrieved
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moresearch.com/dna/plasmid-dna-
purification
Plasmid Isolation (2014). Retrieved February 16,
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?sub=3&brch=77&sim=314&cnt=1
Plasmids (2013). Retrieved February 16, 2014
from http://www.nature.com/scitable/
definition/plasmid-plasmids-28
Restriction Enzymes (2013). Retrieved February
16, 2014 from http://users.rcn.com/jkim
ball.ma.ultranet/BiologyPages/R/Restricti
onEnzymes.html
Rhodes, G., et al. (2004). Complete Nucleotide
Sequence of the Conjugative Tetracycline
Resistance Plasmid pFBAOT6, a Member
of a Group of IncU Plasmids with Global
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Microbiology 70, 7497-7510
Rohde, C. & Henze, B. (2011). Plasmid Isolation
from Bacteria. Retrieved February 16,
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gshinweise/Plasmid_Isolation_from_Bact
eria.pdf
Sambrook J. and D. Russell. (2001). Plasmids
and their usefulness in molecular cloning.
In:Molecular Cloning: A Laboratory
Manual, Vol.1 , 3
rd
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Spring Harbor, NY. P. 1.2-1.29
Stryer, L., Berg, J. & Tymoczko, J. (2002).
Restriction Enzymes: Performing Highly
Specific DNA-Cleavage Reactions, in
Biochemistry (5
th
ed.). New York: W. H.
Freeman
Troubleshooting Guide for DNA Digestion (2014).
Fermentas Life Sciences
Types of Restriction Endonucleases (2014).
Retrieved February 7, 2014 from
https://www.neb.com/products/restriction-
endonucleases/restriction-
endonucleases/ types-of-restriction-
endonucleases
What is restriction enzyme star activity? (2014).
Retrieved February 8, 2014 from
http://worldwide.promega.com/resources/
Biochem 121.1 Plasmid DNA Extraction and Restriction Enzyme Digestion Page 8 of 8
pubhub/enotes/what-is-restriction-
enzyme-star-activity


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2010-00269


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2010-29246


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2010-48759