Restriction Digestion of Lambda DNA: Instruction

Application Note L-01
Restriction Digestion of Lambda DNA
Aim: To investigate the efficiency and outcome of cutting single-digested lambda-DNA

with the restriction enzyme EcoRI, using Wealtecs CB-1 Block Cooler as incubation system.
EcoRI is a Type II restriction enzyme, isolated from E coli, that recognises and cuts the
sequence:
G|AATT C
C TTAA|G
INSTRUCTION
Classification: Restriction enzymes, or restriction endonucleases, are proteins that

recognize and cleave specific sequences of double stranded DNA (Mani et al., 2005a). Most,
but not all of the 3000 discovered restriction enzymes found so far, come from bacteria,
where they serve as a protection system, defending bacteria from foreign DNA.
Restriction enzymes are divided into three major groups based on different preferences
associated with recognition/cutting-sites and environmental requirements, e.g. certain
temperature, ionic strength and pH. Type I and III restriction enzymes recognise certain
DNA sequences and cut some distance away, some times as far as 10 000 base pairs away,
whereas Type II restriction enzymes cut sequences located at a close proximity to the
recognition site. Most restriction enzymes recognise sequences that are relatively short,
often 4-8 base pairs in length. Furthermore, Type II enzymes, which comprise the most
abundant group of enzymes, often recognise palindrome sequences and cut within or
adjacent to these sequences.
Applications: The discovery of restriction enzymes in the late 1960s has had an enormous
impact on molecular biology research. Cutting large DNA-molecules with restriction
enzymes makes it possible to purify homogenous DNA-fragments of defined lengths that
can be subsequently enzymatically manipulated and analysed (Mani et al., 2005a). Cut
DNA-fragments can easily be ligated into DNA-molecules with corresponding ends,
thereby making recombinant DNA-molecules. Due to the exact specificity of restriction
enzymes, and the specific cleavage patterns generated when cut DNA is run on gels,
restriction enzymes also enables for mapping of DNA. A common application of this is
restriction fragment length polymorphism (RFLP), used for e.g. paternity testing. In RFLP,
specific human genomic DNA-areas are cut by several restriction enzymes, and the
fragments are subjected to electrophoresis. The generated fragment pattern is unique for
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a given individual, but shares certain similarities with patterns generated by related
individuals. Moreover, restriction enzymes can also be used to detect specific
variations/mutations in DNA caused by a single nucleotide change, so called single
nucleotide polymorphisms (SNPs) (Dear 2005). The SNP may generate a new cutting site,
or it may result in the loss of a cutting site. Therefore, by cutting DNA with restriction
enzymes directed at the site of a possible mutation and thereafter subjecting the
fragments to electrophoresis, the gain or loss of a band tells whether a SNP is present or
not.
Working conditions: Finding the optimal operating condition for restriction enzymes can
be a hassle. pH and salt-concentration is very important, which is why companies selling
restriction enzymes frequently also provide the appropriate buffer compatible with the
enzyme. Some enzymes are very sensitive to certain ions, such as potassium or sodium,
2+
whereas others work in a wide range of ionic strengths. The divalent cat ion Mg is
required by most restriction enzymes. The correct working temperature is also important,
simply because the bacteria that originally house the restriction enzyme have different
temperature ranges within which they operate optimally. Many bacteria have operation
optima around body temperature (37°C), but some prefer extreme heat or cool
temperatures.
Figure 1: Wealtec’s CB-1 Block Cooler. With a temperature range from 0 to 70°, CB-1 covers a wide range of
incubation applications within fields of molecular biology. CB-1 has 9 pre-set temperatures and customised
temperature setting is extremely easy. An insulated lid and exact temperature control makes CB-1 perfect
for e.g. restriction digestion applications.
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MATERIALS AND METHODS
A restriction mixture consisting of lambda DNA, completely digested by HindIII (Wealtec,

Taipei, Taiwan), EcoRI restriction enzyme (10 U/μl; Fermentas, Glen Blunie, MD, U.S.A.),
10 X EcoRI digestion buffer (Fermentas), and nuclease free water (New England Biolabs,
Ipswich, MA, U.S.A) was added to eppendorf tubes to a final volume of 20 μl for incubation
at 37°C (pre-setting) for one hour in the block cooler (Wealtec).
In order to guarantee the correct incubation temperature, the block cooler had been
pre-warmed for 30 minutes, before loading the samples. A 0.8 % agarose gel was cast
using agarose (Wealtec) and the GES-system with casting tray, comb, dams-claws and
electrophoresis tank (Wealtec). Gels were allowed to set for at least one hour before
loading samples. Two different DNA-ladders (500 bp DNA-ladder; Bangalore Genei,
Bangladore, India and 1 kb DNA-marker; New England Biolabs), were loaded at each sides
of the single and double digested DNA. The DNA was resolved on the gel at 120 V for
approximately 70 minutes. Thereafter the gel was soaked in ethidium bromide solution (5
μg/ml; BioRad), for 30 minutes. After a brief soak in ddH 20, the gel was visualised using
Dolphin-Doc Plus Image system (Wealtec).
RESULT
Figure 2 A and B: Normal (A) and inverted (B) images of restriction digests from single and double digested
lambda-DNA, run on a 0.8 % agarose gel. Lane 1: 500 bp DNA-ladder (0.2 μg), lane 2: lambda- DNA cut by
HindIII (0.5 μg), Lane 3, 4 and 5: lambda-DNA cut by HindIII and EcoRI(all 0.5μg), Lane 6: 1 kb
DNA-ladder(0.2 μg).
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DISCUSSION
Figure 2 shows the cutting pattern of single digested lambda-DNA cut by HindIII in lane 2,
and double digested DNA cut by HindIII and EcoRI in lanes 3, 4 and 5.
Larger DNA-fragements have been cut into smaller, appearing as new bands further down
in the gel. The digestion appears to have been working under current circumstances;
however, there may be several reasons why the reaction sometimes fails. Some
restriction enzymes will alter recognition specificity and/or efficiency when operating
under sub-optimal conditions, a phenomenon called “star”-activity (Mani et al., 2005b).
The reasons for star-activity can be e.g. a high content of glycerol, low salt strength, too
2+
high pH or enzyme concentration, absence of Mg or traces of organic solvents, such as
phenol. As an example, EcoRI, which under optimal circumstances cleaves at G|AATTC,
will instead cleave at N|AATTN under high pH and low ionic strength, where N represents
any nucleotide. Another factor that might impair restriction cutting even though the
conditions are correct is methylation of DNA. If the target sequence of DNA is methylated,
the restriction enzyme will not recognise the site. Methylation is in fact the way bacteria
protect their own DNA from being cut by their own restriction enzyme. Bacteria will
produce not only a restriction enzyme, but also a specific methyl transferase, methylating
those sequences in the bacterial DNA that would be potential targets of the restriction
enzyme.
With a temperature range from 0 to 70°C, Wealtec’s CB-1 Block Cooler allows for multiple
incubation applications in molecular biology. With excellent temperature control, CB-1 is
especially suited for enzymatic reactions such as restriction digests. With cooling
temperatures closer to zero, CB-1 is also perfect for thawing samples and keeping
sensitive enzymes cool.
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REFERENCE
Dear P. H. (2005) Genome Mapping, Encyclopedia of Life Sciences, John Wiley & Sons,
Ltd., www.els.net doi: 10.1038/npg/els.0005353
Mani M, Kandavelou K., Chandrasegaran S., (2005a) Restriction Enzymes, Encyclopedia of

Life Sciences, John Wiley & Sons, Ltd., www.els.net doi: 10.1038/npg/els.0003837
Mani M, Kandavelou K., Wu J., Chandrasegaran S., (2005b) Restriction Enzymes,

Encyclopedia of Life Sciences, John Wiley & Sons, Ltd., www.els.net doi:
10.1038/npg.els.0005332
Tove Sivik Wealtec Bioscience Co. Ltd.

Joy Lee 27Fl. No. 29-1 Sec.2, Jungjeng E. Rd., Danshuei Jen, Taipei, Taiwan 25170
Email: [email protected] TEL: +886-2-8809-8587 FAX: +886-2-8809-8589; http://www.wealtec.com
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Application Note L-01

Restriction Digestion of Lambda DNA

Aim: To investigate the efficiency and outcome of cutting single-digested lambda-DNA

Classification: Restriction enzymes, or restriction endonucleases, are proteins that

A restriction mixture consisting of lambda DNA, completely digested by HindIII (Wealtec,

Mani M, Kandavelou K., Chandrasegaran S., (2005a) Restriction Enzymes, Encyclopedia of

Mani M, Kandavelou K., Wu J., Chandrasegaran S., (2005b) Restriction Enzymes,

Tove Sivik Wealtec Bioscience Co. Ltd.

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