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An Improved Alkaline Lysis

Method for Minipreparation
of Plasmid Dna

Article in Preparative Biochemistry & Biotechnology · March 1999

DOI: 10.1080/10826069908544692 · Source: PubMed

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An Improved Alkaline Lysis

Method for Minipreparation
of Plasmid Dna
J. T. Liou , B. H. Shieh , S. W. Chen & C. Li
a
Institute of Molecular Medicine
b
Institute of Biochemistry National Cheng Kung
University Medical College 1 , University Road
Tainan 70701, Taiwan, ROC
Published online: 04 Mar 2008.

To cite this article: J. T. Liou , B. H. Shieh , S. W. Chen & C. Li (1999) An

Improved Alkaline Lysis Method for Minipreparation of Plasmid Dna, Preparative
Biochemistry and Biotechnology, 29:1, 49-54, DOI: 10.1080/10826069908544692

To link to this article: http://dx.doi.org/10.1080/10826069908544692

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 PREP. BIOCHEM. & BIOTECHNOL., 29(1), 49-54 (1999)
Downloaded by [University of Colorado - Health Science Library] at 11:14 04 September 2014

AN IMPROVED ALKALINE LYSIS METHOD FOR

MINIPREPARATTON OF PLASMID DNA
J. T. Liou,1 B. H. Shieh,1 S. W. Chen,2 C. Li1
1
Institute of Molecular Medicine
2
Institute of Biochemistry
National Cheng Kung University Medical College
1, University Road
Tainan 70701, Taiwan, ROC

ABSTRACT

This study is to improve the digestion pattern of miniprepped plasmid

analyzed on gel. Frequently, some ambiguous DNA bands, which are
suspected to be denatured DNA molecules, appear during electrophoresis
of enzyme digested miniprepped plasmids. By employing Southern
hybridization of two identical gels, one had been treated with
denaruration-neutralization step and another without such treatment, we
confirmed that many of these ambiguous DNA bands were single-
stranded (SS) DNA molecules.

The presence of SS DNA was due to the use of excess amount of

NaOH during plasmid DNA purification with the conventional alkaline
lysis method. We, therefore, modified the procedure and recommend
that a half amount of NaOH (0.1N instead of 0.2N) should be used when
isolating small quantity of plasmid DNA with the method.

49

Copyright © 1999 by Marcel Dekker, Inc. www.dekker.com

 50 LIOUETAL.

INTRODUCTION

Recombinant DNA technology has been routinely used in a variety of

research fields to clone genes of interest. Small scale of recombinant
covalently-closed-circular (plasmid) DNA purification or minipreparation are
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frequently performed during the process. However, we and other colleagues

often detect some ambiguous DNA bands, additional to the vector and DNA
inserts, during minipreparation of plasmid DNA with the most commonly used
procedure, the conventional alkaline lysis method,1'3 followed by restriction
enzyme digestion and gel electrophoresis (Fig. 1A). Appearances of such
ambiguous DNA molecules, in the most cases, are obstacles for analyzing the
results produced by gel electrophoresis.

The conventional alkaline lysis method consists of three consecutive

treatments, i.e., bacterial cell resuspension, NaOH/sodium dodecyl sulfate (SDS)
lysis, and potassium acetate (or sometimes sodium acetate) precipitation steps.1"3
It has been documented that plasmid DNA could be denatured if prolonged
exposure to alkali during the purification process with the alkaline lysis
method.'"3'4 Consequently, the denatured plasmid DNA resisted restriction
enzyme digestion. However, these experiments were not performed with
minipreped plasmid DNA per se and, thus, the available data provided very
limited information for us to identify denatured DNA molecules on gel and
determine the physical nature of those ambiguous DNA bands we detected here
(Fig. 1A). To solve this problem, we characterized the ambiguous DNA bands
by employing the Southern hybridization technique.5

EXPERIMENTAL

Plasmid vector pBluescript (BS; Stratagene, La Jolla, CA, USA; all

molecular biology reagents used here were purchased from Stratagene unless
specified) was first digested with BamHI and ligated to Sau3AI digested human
ILF cDNA.6 The ligation mixture was then transformed into E. cjjli strain
JM109, XL1 Blue, and DH5a (Gibco BRL, Gaithersburg, MD, USA)
individually. The recombinant bacterial colonies were grown in 1.5 mL medium
followed by minipreparation of plasmid DNA with the conventional alkaline
lysis method.2"3 The purified plasmids were then digested with restriction
enzymes Xbal and PstI to separate the vector ("BS" in Fig 1) and insert ("Insert"
in Fig. 1A) DNA on gel.

RESULTS AND DISCUSSION

Fig. 1A illustrates that, in addition to the BS and insert DNA, many

ambiguous DNA bands were also detected on the gel and some of them (marked
 MINIPREPARATION OF PLASMID DNA 51

A.
M
Wl 2 3 4 5
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BS

Inserts

B.
1 2 3 4 5 6 7 8 9 10
4.507 —
2.838
BS

Figure I. Detection of SS DNA bands during electrophoresis of the restriction enzyme

digested miniprepped plasmid DNA. (A) Five plasmids in JM109 cells were isolated
with the conventional alkaline lysis method as previously described.2'5 Two nL of these
DNA solutions were digested with PstI and Xbal for separating vector (indicated as
"BS") and insert (marked "Inserts") DNA on the gel. Agarose gel electrophoresis of the
restricted plasmid DNA was performed according to the procedure described earlier.7
Plasmids on lanes 1,4, and 5 contain linear BS vector as well as insert DNA fragments.
Whereas lanes 2 and 3 separated a digested plasmid without insert and a linear
recombinant plasmid that lost one of two restriction enzyme' sites, respectively.
Molecular standard (MW) used here were bacteriophage X DNA digested with PstI. (B)
Two identical gels as in Fig. 1A were blotted onto nitrocellulose membranes for Southern
hybridization5 with 32P-labeled BS probe. Briefly, gel on left (lanes 1 to 5) was soaked in
20X SSC (3M NaCl and 0.3 M Na3Citrate) immediately before blotting, whereas gel on
right (lanes 6 to 10) was treated with the denaturation and the neutralization solutions
before transferring to membrane. Labeling of BS was performed with random primed
labeling kit (Boehringer Mannheim, Indianapolis, IN, USA) and a-32P dCTP (Amersham,
Little Chalfont, Buckinghamshire, UK). Approximately 105 cpm of the labeled DNA
probe per mL of hybridization solution was used. All DNA bands, except insert DNA, on
the left gel (lanes 6 to 10) hybridized to the BS probe, because they had been denatured.
Whereas only SS DNA bands, without prior denaturation treatment, were capable of
hybridizing to the probe (the right panel; lanes 1 to 5).
 52 LIOUETAL.

by white arrows) behaved just like single:stranded (SS) DNA molecules

(appeared more diffuse and bound less etnidium bromide) as determined
previously.3'7 Production of this confused digestion pattern of miniprepped
plasmid DNA was independent of E. £Qli strains used (data not shown).
Subsequently, two identical gels as in Fig. 1A were blotted onto nitrocellulose
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membranes for Southern hybridization with 32P-labeled BS probe. Fig. I B .

shows that all DNA bands containing the vector sequence on lanes 6 to 10
hybridized to BS probe because the gel had been treated with denaturation-
neutralization step before blotting.7 Whereas only SS DNA molecules (black
arrows on lanes 1 to 5 of Fig. IB), which were identical to those DNA bands
marked by white arrows in Fig. 1A, were able to hybridize to the labeled probe
without a prior denaturation step. Furthermore, these denatured DNA bands,
marked by black arrows in Fig. IB, were already detected in the plasmid DNA
minipreparations without restriction enzyme digestion, when analyzed with
agarose gel electrophoresis (data not shown).

Taken together, we confirmed that the ambiguous bands marked by white

arrows in Fig. 1A were SS plasmid DNA that resisted enzyme digestion,
presumably caused by a prolonged treatment with alkali during purification
process. As for the DNA bands located above the BS vector on lanes 1, 4, and
5, they could be, relaxed, nicked, or incompletely digested recombinant double-
stranded (DS) DNA molecules, as characterized by the hybridization experiment
and the previous report.1

To improve the quality of miniprepped plasmid DNA for routine digestion

analysis on gel, we investigated the effects of alkaline reagent used on
miniprepped plasmid DNA in detail. We found that the shorter alkali (the
alkaline lysis buffer is 0.2N NaOH and 1% SDS) incubation time from regular
five minutes to less than 10 seconds on ice or using less SDS did not improve
the situation at all (data not shown). On the contrary, when a half or less amount
of NaOH (0.1N or less; the amount of SDS remained 1%) was applied to the
same procedure, those denatured plasmid DNA bands were diminished (Figure
2). We, therefore, concluded that excess NaOH in the lysis solution of the
conventional alkaline lysis method caused the denaturation of DS plasmid DNA.
Certain commercial available mini-quantity DNA preparation kits provide users
a stronger buffer (50 mM Tris-HCl, pH 8.0 instead of 25 mM) for resuspending
bacterial cells, the first step in the alkaline lysis method. However, this
modification did not greatly affect, if at all, pH of cell lysates during the two
subsequent treatments and thus the denatured DNA molecule remained detected
on gel (data not shown).

Another protocol has pointed out that glucose in the cell resuspending
solution serves as a buffer during addition the treatment with alkali in the
subsequent step.8 We routinely used 50mM glucose in all experiments
performed here but denaturation of plasmid DNA remained detected, as long as
 MINIPREPARATION OF PLASMID DNA 53

M
Wl 23456789101112
BS
1,700
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1159
805 ^H^^^^^^^H-I
|J Inserts
NaOH
Cone

Figure 2. Minipreparations of plasmid DNA with decreased amounts of NaOH.

Minipreped plamid DNA on lanes 1, 2, 4 of Fig. 1A were used in this experiment.
During mini-plasmid DNA purification with the alkaline lysis method,2-3 decreasing
amounts of NaOH were used (0.2N on lanes 1, 5, and 9; 0.1N on lanes 2, 6, and 10; 0.05
N on lanes 3,7, and 11; 0.02N on lanes 4, 8, and 12). Two uL of the purified minipreped
DNA solutions were, then, subjected to enzyme digestion as described in the Fig. 1.
Denatured DNA (marked by black arrows) could only be detected when regular amount
of NaOH (0.2N) was used to treat cells (lanes 1, 3, and 4). Whereas NaOH
concentrations equal or less than 0.1N did not produce denatured plasmid DNA. The
yields of minipreped plasmid DNA remained the same in all samples although less NaOH
was used (data not shown). However, less NaOH did cause inefficient degradation of
small molecular weight RNA, as shown at the bottom of the gel, suggests that both
RNaseA and alkali contributed to the process. Molecular standard (MW) used here were
bacteriophage X DNA digested with Pstl.

0.2N NaOH was applied (Fig. 1 and 2). Like the prior experiments, addition of
glucose to the cell resuspending solution did not affect pH of lysates either
(data not shown). Therefore, the buffering capacities of these modified
protocols were not strong enough to keep plasmid DNA from denaturation by
the subsequent NaOH treatments. We believe that to effectively protect plasmid
DNA from denaturation, lesser alkali should be used.

CONCLUSIONS

In summary, this study demonstrated that use of 0.1N instead of the regular
amount of NaOH (0.2N) in the lysis solution gave the best results regarding to
the reduction of unwanted DNA molecules and sufficient removal of small
molecular weight RNA (Fig. 2).

We, therefore, recommend that 0.1N NaOH should be applied if the

conventional alkaline lysis method is chosen to purify plasmid DNA in small
quantities.
 54 LIOUETAL.

ACKNOWLEDGMENTS

We thank Dr. Chih-Li Lilian Hsu, for discussions and critical readings of
the manuscript and Yun-Er Liau, and Yao-Pei Yan for preparing the materials
and photographs for publication. This study is a part of the on-going project
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supported by a grant (NSC87-2312-B006-002) to C.L.

Corresponding author of this study is C. Li, Ph.D., Institute of Molecular

Medicine, National Cheng Kung University Medical College, Number 1,
University Rd., Tainan 70701, Taiwan (Office: 886-6-2353535 ext. 3652; FAX:
886-6-2095845; e-mail: chinglifgmail.ncku.edu.twV

Current address for Dr. B. H. Shieh is Department of Biochemistry, Chung

Shan Medical and Dental College, number 113, Sec. 2, Ta-Chien St., Taichung
40203, Taiwan.

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(1988).

3. J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory

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4. J. Vinograd, J. Lebowitz, J. Gen. Physiol., 49,103-125 (1966).

5. E. M. Southern, J. Mol. Biol., 98, 503-517 (1975).

6. C. Li, A. J. Lusis, R. Sparkes, A. Nirula, R. Gaynor, Genomics, 13, 665-671

(1992).

7. C. Li, B.-H. Shieh, I.-H. Su, W.-O. Chuang, BioTech., 24, 60-64 (1998).

8. J. S. Heilig, "Large-Scale Preparation of Plasmid DNA," in Current

Protocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston,
D. D. Moore, eds., John Wiley & Sons, Inc., New York, 1994, Unit 1.7.

Received August 27,1998

Accepted September 15,1998
Manuscript 7031