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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Extraction of Nucleic Acids from Agarose Gel -A Quantitative and Qualitative Comparison of Four Different Methods a
K. K. Pun & W. Kam
a
a
Department of Medicine, Laboratory Medicine Biophysics and Biochemistry , University of California , San Francisco S.F., CA, 94123, U.S.A. Published online: 23 Oct 2006.
To cite this article: K. K. Pun & W. Kam (1990) Extraction of Nucleic Acids from Agarose Gel -A Quantitative and Qualitative Comparison of Four Different Methods, Preparative Biochemistry, 20:2, 123-135, DOI: 10.1080/00327489008050184 To link to this article: http://dx.doi.org/10.1080/00327489008050184
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Tulane University] at 10:05 19 October 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-andconditions
PREPARATIVE BIOCHEMISTRY, 20(2), 123-135 (1990)
Downloaded by [Tulane University] at 10:05 19 October 2014
EXTRACTION OF NUCLEIC ACIDS FROM AGAROSE GEL A QUANTITATIVE AND QUALITATIVE COMPARISON OF FOUR DIFFERENT METHODS
-
K. K. Pun and W. Kam Department o f Medicine, Laboratory Medicine, Biophysics and Biochemistry, U n i v e r s i t y o f C a l i f o r n i a , San Francisco, S.F., CA 94123, U.S.A. ABSTRACT Agarose gel electrophoresis i s commonly used t o separate d i f f e r e n t species o f n u c l e i c acids. We compare f o u r d i f f e r e n t methods o f e x t r a c t i o n which are commonly used. These methods i n c l u d e b u f f e r e x t r a c t i o n , e l e c t r o e l u t i o n , g l a s s bead e x t r a c t i o n and e x t r a c t i o n o f DNA from low-melting agarose. The r e s u l t s show t h a t DNA e x t r a c t e d by these f o u r methods i s comparable i n t h e i r l i g a b i l i t y t o t h e PMT 2 1 vectors and t h e plasmids w i t h i n s e r t can be used f o r subsequent t r a n s f e c t i o n s o f competent b a c t e r i a . There i s a higher y i e l d f o r b u f f e r e x t r a c t i o n and e l e c t r o e l u t i o n when compared w i t h glass bead e x t r a c t i o n and low m e l t i n g agarose (pt0.05). To conclude, t h e f o u r commonly used methods f o r DNA i s o l a t i o n are comparable q u a l i t a t i v e l y . But t h e simplest method, namely b u f f e r e x t r a c t i o n , has t h e highest y i e l d . INTRODUCTION
Agarose gel electrophoresis i s an indispensable technique i n recombinant DNA technology'. It i s valuable both as an a n a l y t i c a l procedure and as a p r e p a r a t i v e process f o r i s o l a t i n g i n d i v i d u a l species o f DNA molecules from a mixture2. Several methods f o r recovery o f DNA from g e l s are commonly used. However each method has i t s own advantages and disadvantages and no study has been performed t o compare t h e various methods. Thus i t i s d i f f i c u l t t o choose t h e best method f o r use i n t h e l a b o r a t o r y 3 . A major problem i n t h e p r e p a r a t i v e use i s t o recover DNA from i n d i v i d u a l g e l band i n good y i e l d and y e t f r e e o f contamination by agarose 123 Copyright 0 1990 by Marcel Dekker, Inc.
124
PUN AND KAM
'.
This study aims a t comparing f o u r commonly used methods of recovery o f DNA from agarose g e l s . These methods i n c l u d e buffer e x t r a c t i o n , e l e c t r o e l u t i on, g l ass bead e x t r a c t i o n and e x t r a c t i o n of
DNA
from low-melting
agarose.
Both q u a n t i t a t i v e
and
q u a l i t a t i v e assessments were performed.
Downloaded by [Tulane University] at 10:05 19 October 2014
MATERIALS AND METHODS
M a t e r i a1 s. corporation.
Sea Kem ME agarose was Marine C o l l o i d s D i v i s i o n .
obtained Bacterial
f r o m FMC a1 k a l i n e
phosphatase, r e s t r i c t i o n enzymes, d i a l y s i s t u b i n g and DNA from bacteriophage were obtained from Bethesda Research Laboratories (Gaithersburg, Maryland). S i l i c a - 3 2 5 mesh (a powdered f l i n t glass) was obtained from l o c a l ceramic shop. A l l o t h e r reagents were purchased from standard 1aboratory suppl i e r s . 600 ug o f DNA f r o m P r e D a r a t i o n o f P z - l a b e l e d DNA. bacteriophage was c u t w i t h t h e r e s t r i c t i o n enzyme, Hind II15. 100 ug o f t h e digested fragments was subsequently P3*-labeled t o a s p e c i f i c a c t i v i t y o f about lo6 cpm/ug by end-labeling w i t h T4
DNA polymerase6. The l a b e l e d fragments were mixed w i t h t h e remaining 500 ug o f non-labeled DNA fragments and separated by agarose gel e l e c t r o p h o r e i s (30 mA overnight). A f t e r e t h i d i u m bromide s t a i n i n g , DNA-containing s l i c e s (each w i t h about 10'cpm) were c u t o u t and subject t o one o f t h e t h r e e d i f f e r e n t e x t r a c t i o n procedures, namely b u f f e r e x t r a c t i o n , e l e c t r o e l u t i o n o r g l a s s bead e x t r a c t i o n . A l i q u o t s w i t h s i m i l a r amount o f r a d i o a c t i v i t y were run on low-melting agarose g e l . various methods were compared.
The r e s u l t s as obtained by
Methods o f DNA recovery from qels. The f o l l o w i n g 4 methods o f e x t r a c t i o n were compared i n t h i s study. The i n d i v i d u a l band o f i n t e r e s t was subject t o e x t r a c t i o n . The f i n a l e x t r a c t e d DNA was assessed both q u a n t i t a t i v e l y and q u a l i t a t i v e l y . Method 1 Recoverv o f DNA from low-me1 t i n a aqarose q e l Electrophoresis o f t h e DNA r e s t r i c t i o n fragments was performed on
.
125
EXTRACTION OF NUCLEIC A C I D S FROM AGAROSE GEL
'.
l o w - m e l t i n g a g a r o s e a t 4 O C as d e s c r i b e d a b o v e The g e l fragments o f i n t e r e s t (0.56, 2.0, 4.4 and 9.5 Kb) were c u t o u t w i t h r a z o r blades. The agarose around each i n d i v i d u a l band was subsequently m e l t e d a t 65'
f o r 5 minutes.
The DNA was t w i c e
e x t r a c t e d w i t h phenol a t room temperature and p r e c i p i t a t e d w i t h
1/10 volume o f 7.5 M ammonium a c e t a t e (pH 7.5) and 2 volumes o f
Downloaded by [Tulane University] at 10:05 19 October 2014
c o l d ethanol
(0
O).
After precipitation,
t h e p r e c i p i t a t e was
d i s s o l v e d i n 20 u l o f b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and 1 mM EDTA8. Method 2 E l u t i o n bv d i f f u s i o n . The procedure c o n s i s t e d o f crushing gel slices i n t o f i n e p a r t i c l e s i n 1 m l o f b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and 1 mM EDTA'.
The DNA fragments
were subsequently e l u t e d by i n c u b a t i o n f o r s e v e r a l hours a t 37OC i n 5 m l o f a b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and 10 mM NaCl . The e l u t e d DNA was p r e c i p i t a t e d as d e s c r i b e d above and t h e n r e d i s s o l v e d i n 20 u l o f b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and
1 mM EDTA. Method 3
E l e c t r o e l u t i o n o f DNA o n t o d i a l y s i s membrane.
was e l e c t r o e l u t e d from g e l s l i c e s by
DNA Small
s l i c e s o f agarose g e l were r i n s e d i n a b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and 1 mM EDTA. Subsequently t h e g e l s l i c e s were p l a c e d i n 18 mm d i a l y s i s t u b e i n t h e same b u f f e r . A r e c t a n g u l a r p l a s t i c box w i t h p l a t i n u m e l e c t r o d e s a l o n g o p p o s i t e s i d e s s e r v e s as t h e e l e c t r o - e l u t i o n chamber. DNA was e l e c t r o e l u t e d o u t o f t h e g e l and a g a i n s t t h e w a l l s o f t h e sack by e l e c t r o p h o r e s i s w i t h d i r e c t c u r r e n t f o r about 1/2 hour, and t h e p o l a r i t y was r e v e r s e d f o r about 10 seconds t o d e t a c h t h e DNA f r o m t h e s u r f a c e o f t h e membrane. The b u f f e r was t h e n p i p e t t e d f r o m t h e bag and t h e DNA p r e c i p i t a t e d as d e s c r i b e d above. Method 4 Glass Bead E x t r a c t i o n .
The g e l was ground up and
d i s s o l v e d i n a s o l u t i o n c o n t a i n i n g 2 mg/ml sodium i o d i d e a t 37
OC
f o r 2 hours l 3 9 l 4 . 40 u l o f g l a s s powder were added t o t h e s o l u t i o n a t '0 C t o l e t t h e DNA adsorb o n t o g l a s s o v e r n i g h t . 20
126
PUN AND KAM
u l o f t h e s l u r r y was shown t o be more than enough f o r adsorption o f DNA f r o m one s l i c e ( d a t a n o t shown). A f t e r o v e r n i g h t incubation, t h e glass p e l l e t w i t h t h e adsorbed DNA was spun down i n microfuge and subsequently washed w i t h 10 volumes o f t h e same sodium i o d i d e s o l u t i o n . The p e l l e t s were then e l u t e d i n 3 volumes o f a b u f f e r c o n t a i n i n g 50 mM T r i s (pH9) and 0.2 M NaCl a t 60' C
Downloaded by [Tulane University] at 10:05 19 October 2014
f o r 30 minutes. The e x t r a c t i o n was repeated once. The two e x t r a c t i o n s were p o o l e d and p r e c i p i t a t e d w i t h e t h a n o l . The p r e c i p i t a t e was r e c o n s t i t u t e d i n 20 u l b u f f e r c o n t a i n i n g 1 mM Tris
(pH7.5)
and 0.1
mM EDTA and
s u b s e q u e n t l y used f o r
q u a n t i t a t i o n o f recovery.
-
A t e s t o f aualitv. L i q a t i o n and subcloninq o f e x t r a c t e d DNA The q u a l i t y o f t h e e l u t e d DNA was assessed by i t s a b i l i t y t o serve as substrates f o r DNA l i g a s e . Plasmid pMT 21, was used as t h e vector. The l i g a t i o n r e a c t i o n was performed i n t h e presence o f T4 l i g a s e , 1mM spermidine, 7 mM MgC12 , 70 uM ATP, 70 mM T r i s HC1 (pH 7.5)
as p r e v i o u s l y d e s c r i b e d 15. The m i x t u r e was
incubated f o r 5 hours a t room temperature and then added t o 150 u l HB 101 competent c e l l s . The t r a n s f e c t e d c e l l s were incubated f o r 1 hour a t 4' C and heat-shocked f o r 2 minutes a t 42' C. 1 ml o f L u r i a b r o t h was added t o a l l o w growth o f t h e b a c t e r i a . A f t e r microfuging f o r 2 minutes, t h e supernatant was removed and t h e c e l l s were resuspended i n 150 u l o f L u r i a b r o t h . The c e l l s were f i n a l l y poured on an L ( a m p i c i l l i n ) p l a t e and dispersed w i t h a glass rod. Thus one L p l a t e would be used f o r each fragment o f DNA prepared by each o f t h e 4 d i f f e r e n t methods. A f t e r i n c u b a t i o n f o r 16 hours a t 3loC, a d i s c r e t e colony was picked up from t h e L p l a t e and grown up i n 5 m l o f LB medium. From t h i s c u l t u r e , an a1 k a l i n e SDS minipreparation was performed t o i s o l a t e t h e plasmid DNA f o r f u r t h e r analysis o f p u r i t y by e l e c t r o p h o r e s i s . The m i n i preparation was performed i n accordance w i t h t h e method described by Brinboim16. B r i e f l y , t r a n s f e c t e d b a c t e r i a grown i n t h e c u l t u r e b r o t h were spun down i n a SM-24 r o t o r a t 5,000 rpm
EXTRACTION OF N U C L E I C A C I D S FROM AGAROSE G E L
f o r 5 minutes.
127
The b a c t e r i a p e l 1e t was subsequently resuspended
i n 200 u l o f i c e c o l d s o l u t i o n c o n t a i n i n g 50 mM glucose, 25mM T r i s (pH 8.0) and 10 mM EDTA. The b a c t e r i a were then l y s e d w i t h a 400 u l o f f r e s h l y prepared s o l u t i o n c o n t a i n i n g 0.2 N NaOH and 1% SDS. This was mixed g e n t l y by i n v e r t i n g t h e tube 3 times and h e l d a t O°C f o r 5 minutes. F i n a l l y 300 u l o f i c e - c o l d 5M sodium acetate (pH 4.8) was added and held a t O°C f o r 15 minutes. A Downloaded by [Tulane University] at 10:05 19 October 2014
curd-like precipitate
would form.
This was c e n t r i f u g e d f o r 2
minutes and t h e supernatant was subsequently e x t r a c t e d by phenol
.
DNA i n t h e supernatant was f i n a l l y p r e c i p i t a t e d by c o l d ethanol and t h e p e l l e t dissolved i n 20 u l o f a s o l u t i o n c o n t a i n i n g 40 mM T r i s - a c e t a t e and 1mM EDTA. The i s o l a t e d plasmid DNA was digested by Hind I 1 1 and an a l i q u o t o f each sample was r u n on agarose gel t o examine f o r t h e q u a l i t y o f DNA i n s e r t i n each preparation. Q u a n t i t a t i o n o f t h e y i e l d o f DNA as obtained by d i f f e r e n t methods
o f extraction. To enable accurate assessment, two d i f f e r e n t methods o f q u a n t i t a t i o n were used. The f i r s t method involved q u a n t i t a t i v e assessment of t h e r a d i o a c t i v i t y c o n t a i n e d i n each g e l band b e f o r e and a f t e r e x t r a c t i o n . The amount o f r a d i o a c t i v i t y recovered i n each band was measured i n a Packard s c i n t i l l a t i o n counter, a f t e r m i x i n g with 5 ml o f scintillation fluid. The second method was t o run t h e f i n a l products o f DNA e x t r a c t i o n on a s i n g l e agarose g e l . Both t h e pre-and p o s t e x t r a c t i o n samples were run on t h e same gel f o r comparison. A f t e r electrophoresis, Southern b l o t t i n g was performed and t h e n i t r o c e l l u l o s e membrane was l a t e r used f o r an a ~ t o r a d i o g r a m ' ~ . The amount o f DNA e x t r a c t e d was revealed by t h e i n t e n s i t y o f each band on t h e f i l m as q u a n t i t a t e d by densitometer. RESULTS
Table I shows t h e recovery o f DNA as measured by t h e r a d i o a c t i v i t y i n t h e gel s l i c e s before and a f t e r e x t r a c t i o n . The
PUN AND KAM
128
Downloaded by [Tulane University] at 10:05 19 October 2014
TABLE
I
E l u t i o n e f f i c i e n c i e s w i t h d i f f e r e n t methods o f DNA e x t r a c t i o n from agarose g e l s . DNA fragments were separated on a 1% agarose g e l . The 0.56, 2.0, 4.4 and 9.5 -kbp fragments were c u t out and t h e s l i c e s (volume o f about 100 u l each) were s u b j e c t t o Methods 1 and 3 a r e d i f f e r e n t methods o f DNA e x t r a c t i o n . associated w i t h s i g n i f i c a n t l y lower y i e l d s when compared w i t h methods 2 and 4 (p
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Extraction of Nucleic Acids from Agarose Gel -A Quantitative and Qualitative Comparison of Four Different Methods a
K. K. Pun & W. Kam
a
a
Department of Medicine, Laboratory Medicine Biophysics and Biochemistry , University of California , San Francisco S.F., CA, 94123, U.S.A. Published online: 23 Oct 2006.
To cite this article: K. K. Pun & W. Kam (1990) Extraction of Nucleic Acids from Agarose Gel -A Quantitative and Qualitative Comparison of Four Different Methods, Preparative Biochemistry, 20:2, 123-135, DOI: 10.1080/00327489008050184 To link to this article: http://dx.doi.org/10.1080/00327489008050184
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Tulane University] at 10:05 19 October 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-andconditions
PREPARATIVE BIOCHEMISTRY, 20(2), 123-135 (1990)
Downloaded by [Tulane University] at 10:05 19 October 2014
EXTRACTION OF NUCLEIC ACIDS FROM AGAROSE GEL A QUANTITATIVE AND QUALITATIVE COMPARISON OF FOUR DIFFERENT METHODS
-
K. K. Pun and W. Kam Department o f Medicine, Laboratory Medicine, Biophysics and Biochemistry, U n i v e r s i t y o f C a l i f o r n i a , San Francisco, S.F., CA 94123, U.S.A. ABSTRACT Agarose gel electrophoresis i s commonly used t o separate d i f f e r e n t species o f n u c l e i c acids. We compare f o u r d i f f e r e n t methods o f e x t r a c t i o n which are commonly used. These methods i n c l u d e b u f f e r e x t r a c t i o n , e l e c t r o e l u t i o n , g l a s s bead e x t r a c t i o n and e x t r a c t i o n o f DNA from low-melting agarose. The r e s u l t s show t h a t DNA e x t r a c t e d by these f o u r methods i s comparable i n t h e i r l i g a b i l i t y t o t h e PMT 2 1 vectors and t h e plasmids w i t h i n s e r t can be used f o r subsequent t r a n s f e c t i o n s o f competent b a c t e r i a . There i s a higher y i e l d f o r b u f f e r e x t r a c t i o n and e l e c t r o e l u t i o n when compared w i t h glass bead e x t r a c t i o n and low m e l t i n g agarose (pt0.05). To conclude, t h e f o u r commonly used methods f o r DNA i s o l a t i o n are comparable q u a l i t a t i v e l y . But t h e simplest method, namely b u f f e r e x t r a c t i o n , has t h e highest y i e l d . INTRODUCTION
Agarose gel electrophoresis i s an indispensable technique i n recombinant DNA technology'. It i s valuable both as an a n a l y t i c a l procedure and as a p r e p a r a t i v e process f o r i s o l a t i n g i n d i v i d u a l species o f DNA molecules from a mixture2. Several methods f o r recovery o f DNA from g e l s are commonly used. However each method has i t s own advantages and disadvantages and no study has been performed t o compare t h e various methods. Thus i t i s d i f f i c u l t t o choose t h e best method f o r use i n t h e l a b o r a t o r y 3 . A major problem i n t h e p r e p a r a t i v e use i s t o recover DNA from i n d i v i d u a l g e l band i n good y i e l d and y e t f r e e o f contamination by agarose 123 Copyright 0 1990 by Marcel Dekker, Inc.
124
PUN AND KAM
'.
This study aims a t comparing f o u r commonly used methods of recovery o f DNA from agarose g e l s . These methods i n c l u d e buffer e x t r a c t i o n , e l e c t r o e l u t i on, g l ass bead e x t r a c t i o n and e x t r a c t i o n of
DNA
from low-melting
agarose.
Both q u a n t i t a t i v e
and
q u a l i t a t i v e assessments were performed.
Downloaded by [Tulane University] at 10:05 19 October 2014
MATERIALS AND METHODS
M a t e r i a1 s. corporation.
Sea Kem ME agarose was Marine C o l l o i d s D i v i s i o n .
obtained Bacterial
f r o m FMC a1 k a l i n e
phosphatase, r e s t r i c t i o n enzymes, d i a l y s i s t u b i n g and DNA from bacteriophage were obtained from Bethesda Research Laboratories (Gaithersburg, Maryland). S i l i c a - 3 2 5 mesh (a powdered f l i n t glass) was obtained from l o c a l ceramic shop. A l l o t h e r reagents were purchased from standard 1aboratory suppl i e r s . 600 ug o f DNA f r o m P r e D a r a t i o n o f P z - l a b e l e d DNA. bacteriophage was c u t w i t h t h e r e s t r i c t i o n enzyme, Hind II15. 100 ug o f t h e digested fragments was subsequently P3*-labeled t o a s p e c i f i c a c t i v i t y o f about lo6 cpm/ug by end-labeling w i t h T4
DNA polymerase6. The l a b e l e d fragments were mixed w i t h t h e remaining 500 ug o f non-labeled DNA fragments and separated by agarose gel e l e c t r o p h o r e i s (30 mA overnight). A f t e r e t h i d i u m bromide s t a i n i n g , DNA-containing s l i c e s (each w i t h about 10'cpm) were c u t o u t and subject t o one o f t h e t h r e e d i f f e r e n t e x t r a c t i o n procedures, namely b u f f e r e x t r a c t i o n , e l e c t r o e l u t i o n o r g l a s s bead e x t r a c t i o n . A l i q u o t s w i t h s i m i l a r amount o f r a d i o a c t i v i t y were run on low-melting agarose g e l . various methods were compared.
The r e s u l t s as obtained by
Methods o f DNA recovery from qels. The f o l l o w i n g 4 methods o f e x t r a c t i o n were compared i n t h i s study. The i n d i v i d u a l band o f i n t e r e s t was subject t o e x t r a c t i o n . The f i n a l e x t r a c t e d DNA was assessed both q u a n t i t a t i v e l y and q u a l i t a t i v e l y . Method 1 Recoverv o f DNA from low-me1 t i n a aqarose q e l Electrophoresis o f t h e DNA r e s t r i c t i o n fragments was performed on
.
125
EXTRACTION OF NUCLEIC A C I D S FROM AGAROSE GEL
'.
l o w - m e l t i n g a g a r o s e a t 4 O C as d e s c r i b e d a b o v e The g e l fragments o f i n t e r e s t (0.56, 2.0, 4.4 and 9.5 Kb) were c u t o u t w i t h r a z o r blades. The agarose around each i n d i v i d u a l band was subsequently m e l t e d a t 65'
f o r 5 minutes.
The DNA was t w i c e
e x t r a c t e d w i t h phenol a t room temperature and p r e c i p i t a t e d w i t h
1/10 volume o f 7.5 M ammonium a c e t a t e (pH 7.5) and 2 volumes o f
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c o l d ethanol
(0
O).
After precipitation,
t h e p r e c i p i t a t e was
d i s s o l v e d i n 20 u l o f b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and 1 mM EDTA8. Method 2 E l u t i o n bv d i f f u s i o n . The procedure c o n s i s t e d o f crushing gel slices i n t o f i n e p a r t i c l e s i n 1 m l o f b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and 1 mM EDTA'.
The DNA fragments
were subsequently e l u t e d by i n c u b a t i o n f o r s e v e r a l hours a t 37OC i n 5 m l o f a b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and 10 mM NaCl . The e l u t e d DNA was p r e c i p i t a t e d as d e s c r i b e d above and t h e n r e d i s s o l v e d i n 20 u l o f b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and
1 mM EDTA. Method 3
E l e c t r o e l u t i o n o f DNA o n t o d i a l y s i s membrane.
was e l e c t r o e l u t e d from g e l s l i c e s by
DNA Small
s l i c e s o f agarose g e l were r i n s e d i n a b u f f e r c o n t a i n i n g 40 mM T r i s - a c e t a t e and 1 mM EDTA. Subsequently t h e g e l s l i c e s were p l a c e d i n 18 mm d i a l y s i s t u b e i n t h e same b u f f e r . A r e c t a n g u l a r p l a s t i c box w i t h p l a t i n u m e l e c t r o d e s a l o n g o p p o s i t e s i d e s s e r v e s as t h e e l e c t r o - e l u t i o n chamber. DNA was e l e c t r o e l u t e d o u t o f t h e g e l and a g a i n s t t h e w a l l s o f t h e sack by e l e c t r o p h o r e s i s w i t h d i r e c t c u r r e n t f o r about 1/2 hour, and t h e p o l a r i t y was r e v e r s e d f o r about 10 seconds t o d e t a c h t h e DNA f r o m t h e s u r f a c e o f t h e membrane. The b u f f e r was t h e n p i p e t t e d f r o m t h e bag and t h e DNA p r e c i p i t a t e d as d e s c r i b e d above. Method 4 Glass Bead E x t r a c t i o n .
The g e l was ground up and
d i s s o l v e d i n a s o l u t i o n c o n t a i n i n g 2 mg/ml sodium i o d i d e a t 37
OC
f o r 2 hours l 3 9 l 4 . 40 u l o f g l a s s powder were added t o t h e s o l u t i o n a t '0 C t o l e t t h e DNA adsorb o n t o g l a s s o v e r n i g h t . 20
126
PUN AND KAM
u l o f t h e s l u r r y was shown t o be more than enough f o r adsorption o f DNA f r o m one s l i c e ( d a t a n o t shown). A f t e r o v e r n i g h t incubation, t h e glass p e l l e t w i t h t h e adsorbed DNA was spun down i n microfuge and subsequently washed w i t h 10 volumes o f t h e same sodium i o d i d e s o l u t i o n . The p e l l e t s were then e l u t e d i n 3 volumes o f a b u f f e r c o n t a i n i n g 50 mM T r i s (pH9) and 0.2 M NaCl a t 60' C
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f o r 30 minutes. The e x t r a c t i o n was repeated once. The two e x t r a c t i o n s were p o o l e d and p r e c i p i t a t e d w i t h e t h a n o l . The p r e c i p i t a t e was r e c o n s t i t u t e d i n 20 u l b u f f e r c o n t a i n i n g 1 mM Tris
(pH7.5)
and 0.1
mM EDTA and
s u b s e q u e n t l y used f o r
q u a n t i t a t i o n o f recovery.
-
A t e s t o f aualitv. L i q a t i o n and subcloninq o f e x t r a c t e d DNA The q u a l i t y o f t h e e l u t e d DNA was assessed by i t s a b i l i t y t o serve as substrates f o r DNA l i g a s e . Plasmid pMT 21, was used as t h e vector. The l i g a t i o n r e a c t i o n was performed i n t h e presence o f T4 l i g a s e , 1mM spermidine, 7 mM MgC12 , 70 uM ATP, 70 mM T r i s HC1 (pH 7.5)
as p r e v i o u s l y d e s c r i b e d 15. The m i x t u r e was
incubated f o r 5 hours a t room temperature and then added t o 150 u l HB 101 competent c e l l s . The t r a n s f e c t e d c e l l s were incubated f o r 1 hour a t 4' C and heat-shocked f o r 2 minutes a t 42' C. 1 ml o f L u r i a b r o t h was added t o a l l o w growth o f t h e b a c t e r i a . A f t e r microfuging f o r 2 minutes, t h e supernatant was removed and t h e c e l l s were resuspended i n 150 u l o f L u r i a b r o t h . The c e l l s were f i n a l l y poured on an L ( a m p i c i l l i n ) p l a t e and dispersed w i t h a glass rod. Thus one L p l a t e would be used f o r each fragment o f DNA prepared by each o f t h e 4 d i f f e r e n t methods. A f t e r i n c u b a t i o n f o r 16 hours a t 3loC, a d i s c r e t e colony was picked up from t h e L p l a t e and grown up i n 5 m l o f LB medium. From t h i s c u l t u r e , an a1 k a l i n e SDS minipreparation was performed t o i s o l a t e t h e plasmid DNA f o r f u r t h e r analysis o f p u r i t y by e l e c t r o p h o r e s i s . The m i n i preparation was performed i n accordance w i t h t h e method described by Brinboim16. B r i e f l y , t r a n s f e c t e d b a c t e r i a grown i n t h e c u l t u r e b r o t h were spun down i n a SM-24 r o t o r a t 5,000 rpm
EXTRACTION OF N U C L E I C A C I D S FROM AGAROSE G E L
f o r 5 minutes.
127
The b a c t e r i a p e l 1e t was subsequently resuspended
i n 200 u l o f i c e c o l d s o l u t i o n c o n t a i n i n g 50 mM glucose, 25mM T r i s (pH 8.0) and 10 mM EDTA. The b a c t e r i a were then l y s e d w i t h a 400 u l o f f r e s h l y prepared s o l u t i o n c o n t a i n i n g 0.2 N NaOH and 1% SDS. This was mixed g e n t l y by i n v e r t i n g t h e tube 3 times and h e l d a t O°C f o r 5 minutes. F i n a l l y 300 u l o f i c e - c o l d 5M sodium acetate (pH 4.8) was added and held a t O°C f o r 15 minutes. A Downloaded by [Tulane University] at 10:05 19 October 2014
curd-like precipitate
would form.
This was c e n t r i f u g e d f o r 2
minutes and t h e supernatant was subsequently e x t r a c t e d by phenol
.
DNA i n t h e supernatant was f i n a l l y p r e c i p i t a t e d by c o l d ethanol and t h e p e l l e t dissolved i n 20 u l o f a s o l u t i o n c o n t a i n i n g 40 mM T r i s - a c e t a t e and 1mM EDTA. The i s o l a t e d plasmid DNA was digested by Hind I 1 1 and an a l i q u o t o f each sample was r u n on agarose gel t o examine f o r t h e q u a l i t y o f DNA i n s e r t i n each preparation. Q u a n t i t a t i o n o f t h e y i e l d o f DNA as obtained by d i f f e r e n t methods
o f extraction. To enable accurate assessment, two d i f f e r e n t methods o f q u a n t i t a t i o n were used. The f i r s t method involved q u a n t i t a t i v e assessment of t h e r a d i o a c t i v i t y c o n t a i n e d i n each g e l band b e f o r e and a f t e r e x t r a c t i o n . The amount o f r a d i o a c t i v i t y recovered i n each band was measured i n a Packard s c i n t i l l a t i o n counter, a f t e r m i x i n g with 5 ml o f scintillation fluid. The second method was t o run t h e f i n a l products o f DNA e x t r a c t i o n on a s i n g l e agarose g e l . Both t h e pre-and p o s t e x t r a c t i o n samples were run on t h e same gel f o r comparison. A f t e r electrophoresis, Southern b l o t t i n g was performed and t h e n i t r o c e l l u l o s e membrane was l a t e r used f o r an a ~ t o r a d i o g r a m ' ~ . The amount o f DNA e x t r a c t e d was revealed by t h e i n t e n s i t y o f each band on t h e f i l m as q u a n t i t a t e d by densitometer. RESULTS
Table I shows t h e recovery o f DNA as measured by t h e r a d i o a c t i v i t y i n t h e gel s l i c e s before and a f t e r e x t r a c t i o n . The
PUN AND KAM
128
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TABLE
I
E l u t i o n e f f i c i e n c i e s w i t h d i f f e r e n t methods o f DNA e x t r a c t i o n from agarose g e l s . DNA fragments were separated on a 1% agarose g e l . The 0.56, 2.0, 4.4 and 9.5 -kbp fragments were c u t out and t h e s l i c e s (volume o f about 100 u l each) were s u b j e c t t o Methods 1 and 3 a r e d i f f e r e n t methods o f DNA e x t r a c t i o n . associated w i t h s i g n i f i c a n t l y lower y i e l d s when compared w i t h methods 2 and 4 (p
