Volume 5 Number 3 March 1978
Nucleic Acids Research
Separation of very large DNA molecules by gel electrophoresis Walton L. Fangman Department of Genetics, SK-50, University of Washington,Seattle, WA 98195, USA
Received 16 December 1977 ABSTRACT Very large DNA molecules were separated by electrophoresis in horizontal slab gels of dilute agarose. Conditions of electrophoresis were developed using intact DNA molecules from the bacterial viruses A, T4 and G. Their DNAs have molecular weights (M) of 32 million, 120 million, and 500 million, respectively. Several electrophoresis conditions were found which give sufficiently high mobilities and large mobility differences that these DNAs are separated in a short time. Electrophoresis in 0.1% agarose at 2.5 V/cm of gel length separates T4 and X DNAs by 2.0 cm, and G and T4 DNAs by 1.0 cm in only 10 hr. With some conditions DNA mobilities are directly proportional to log M for M values from 10 to 500 million. The procedures used will allow rapid molecular weight determination and separation of very large DNA molecules. INTRODUCTION
The DNA molecules of eukaryotic chromosomes, chloroplasts and large viruses have molecular weights of 100 million and greater. Such large DNA molecules cannot easily be separated on the basis of size. Separation by sedimentation velocity suffers from zone spreading which probably results
from convection, and from the necessity of centrifuging for long periods at low centrifuge speed. At high centrifuge speeds DNA molecules exceeding 100 million daltons exhibit a marked reduction in s (see citations in ref. 1). Gel electrophoresis, which has not been applied to very large DNAs, has been extremely useful for separating smaller DNA molecules (less than 10 million daltons). Duplex DNA molecules differing by a few percent in molecular weight can be resolved. A plot of log M versus electrophoretic mobility approximates a straight line with large molecules having a slower rate of migration than smaller ones. However, with the electrophoresis conditions usually employed DNA molecules with a mass above 10 million daltons migrate much faster than expected and are not well resolved. There have been a large number of publications on the electrophoretic behavior of small, less than 25 million dalton, DNA molecules (for citations
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
653
Nucleic Acids Research This paper presents conditions for electrophoretic separation of DNA molecules of 10 to 500 million daltons. The work is an extension of earlier observations of Henckes et al. (ref. 3). Large DNAs employed in developing the electrophoresis conditions were the intact molecules from see ref. 2).
bacterial viruses X, T4 and G. X and T4 DNAs have been well characterized and have masses of 32 million and 120 million daltons, respectively. Bacteriophage G DNA has been reported to have a mass of 500 million daltons (4). DNA was prepared from bacteriophage G particles by procedures which eliminate breakage by shear and used as a molecular weight standard.
MATERIALS AND METHODS Bacteriophage DNA. T4D and XCI857 viruses containing 14C or 3H labels were prepared by standard procedures. Bacteriophage G and its host Bacillus megatherium PGH were obtained from Prof. G. Donelli. The phage was cloned and subsequently titred with beef extract broth (BEB) plates and top agar at 30°. BEB medium contains per liter: 3 g beef extract (BBL), 5 g peptone (Difco), 5 g NaCl, 8 x 105 moles MgSO4, 2 x 10 moles -34 MnSO and 10 moles CaClV final pH 7.1-7.2. Plates contained 10 g agar/l and top agar 7.5 g agar/l. Cells for plating were grown in BEB liquid at
Phage G for DNA isolation was prepared by infecting exponential phase cells in TYEM medium at a culture optical density (660 nm) of 1.5 with 2 x 108 phage per ml of culture. TYEM medium contains per liter: 10 g tryptone (Difco), 5 g NaCl, 0.5 g yeast extract (Difco) and MgSO4, MnSO4, and CaCl2 as in BEB, final pH 7.4-7.5. With vigorous aeration the culture lysed in 3-5 hours. The lysed culture was shaken slowly with chloroform for a few min, then centrifuged at 4,000 x g for 20 min. The supernatant (1-2 x 10 phage/ml) was centrifuged at 27,000 x g for 30 min and the
30°.
pellet containing the phage suspended with cold .OlM Tris, .OlM MgSO4, pH 7.4 (TM) containing 1 mg/ml bovine serum albumin (BSA). This material was incubated with 100 pg/ml RNase I for 15 min at room temperature, then 0.5-1.0 ml (containing 1-5 x 1011 phage) was layered onto a cold 16 ml linear gradient of 10-35% sucrose in TM-BSA. After centrifugation in a Spinco SW27.1 rotor at 14,000 rpm for 30 min (5°), the phage were collected as part of a white diffuse band. DNA was labeled by adding 2.5 pc/ml 6- H-uracil at 0, 1 and 2 hours of infection. DNA was isolated from G particles by very slowly mixing 0.7 ml of phage suspension with 0.7 ml 10% sodium lauroyl sarcosinate in 0.2M EDTA, pH 8 in a Spinco SW50 polyallomer centrifuge tube. The tube was corked
654 t
Nucleic Acids Research and heated at 650 for 10 min.
The solution
underlaid with 3.5 ml
was
temperature-saturated CsCl in 0.2M EDTA, pH 8 and centrifuged in rotor at 30,000 rpm for 2-3 days (100).
Fractions
were
an
room
SW50L
collected through
the tube bottom with a 13G hooded needle (boiled in EDTA solution) at a flow rate of 0.10-0.15 ml/min. The DNA was dialyzed in a Collodion bag (S & S) or dialysis tubing held open at one end with a ring of plastic, against 10 mM Tris, 100 mM EDTA, pH 8 and stored at 5°. For sedimentation analysis
or
gel electrophoresis G DNA
was
kept intact by employing slow
transfers with 1.0-1.5 mm i.d. pipettes. T4 and X DNAs were prepared from virus particles by the same procedure. The initial preparations of X and T4 DNAs used in this work contained intact DNA molecules based on the ratio of their S values and the contour lengths of the DNAs observed by electronmicroscopy (5, 6). Subsequent preparations were characterized by electrophoresis in 0.2% agarose as reported here.
The molecular weight of G DNA
mentation with T4 DNA (Figure 3).
was
determined by cosedi-
H-labeled G DNA (0.20 pg) and
C-
1.0 mM EDTA, pH8, was labeled T4 DNA (0.05 pg) in 0.5 ml 1.0 mM Tris in 0.O1M Tris, sucrose layered on a 58 ml linear gradient of 15 to 30% 0.5M NaCl. The gradient was centrifuged in a 0.2M EDTA, pH8 pH8 Spinco SW25.2 rotor at 8,000 rpm for 3.5 days (5°). Molecular weights -
-
-
(ref. 7). calculated from the equation S1/S2 = (M1/M2) Specific fragments of X DNA were generated with the endonucleases Sal I (New England Biolabs) and Eco RI (supplied by Dr. Maynard Olson and Guy
were
Page). Sizes for X DNA and X DNA fragments were taken from data of Dr. Peter Philippsen (personal communication) obtained from electron microscopic contour length measurements. His kilobase pair values were converted to mass units using 660 daltons/base pair to obtain masses, in millions of daltons, of 32 for intact X DNA, 21 and 10 for the two major Sal I fragments (A and B, respectively), and 14, 4.9, 3.8, 3.6, 3.1 and 2.2 for the six Eco RI fragments (designated here as A, B, C, D, E, and F, respectively). T4 DNA was taken to have a mass of 120 million daltons (8).
Electrophoresis.
Horizontal agarose gels were formed on a glass plate
plastic container with a raised center section separating two buffer reservoirs and electrodes. Buffer was in direct contract with the ends of
in
a
the gel.
Gels were 13.5 cm wide, 10 to 25 cm long and about 1.0 cm thick.
Seakem LE agarose was used for all gels except 0.1% agarose gels in which Seakem HGT(P) agarose was used to provide greater mechanical stability. 655
Nucleic Acids Research Gels consisting of less than 0.4% agarose
1.5
cm
made by first pouring
a
2
agarose were mm
slab, then four sides
This "box" allowed dilute gels
wide.
poured into
to be
picked
a 1
up
"box" of 1% cm
thick and
and moved for
photography and other manipulations. The electrophoresis buffer contained 10.3g Tris, 5.5g boric acid and 0.93g disodium EDTA per liter. For some gels the buffer contained 4.4
g
Tris, 4.1
g
anhydrous monosodium phosphate,
0.37 g disodium EDTA per liter. The two buffers gave only small differences in mobilities. Unless stated otherwise, gels and buffers contained 0.5 Pg/ml ethidium bromide.
Sample wells
ing ten 0.75
mm
x
8.0
in contact with the 1%
teeth. agarose
were
made with
The wells
were
bottom layer.
a
Biorad teflon comb contain-
about 8
mm
deep and
Samples of 30 pl
were
were
not
intro-
a screw-type pipettor and 50 p1 disposable glass micropipettes (1 mm i.d.). Gels of 0.1 and 0.2% agarose were chilled to 50 to stiffen them before removing the comb and
duced into the wells by slow hand pipetting using
loading the wells. The 30 p4 sample was 1 mM Tris, pH 8, 10 mM EDTA, pH 8, 10% glycerol and 0.0015% bromphenol blue and contained 0.05 ig Sal I A DNA fragments, 0.10 vg Eco RI X DNA fragments, and about 0.025 hg each of X DNA, T4 DNA and G DNA. Three-fold lower and three-fold higher amounts of the intact virus DNAs did not result in altered mobilities although streaking toward the wells occurred at the higher concentrations. The mobility of
each DNA run alone was the same as in the mixture. After loading, the entire gel was covered with household plastic wrap and electrophoresis was carried out at room temperature (about 210) at constant voltage. The voltage gradient was measured with a Midland voltmeter using platinum leads at a 10 cm spacing. Gels run without ethidium brosubsequently stained by soaking overnight in buffer containing
inserted into the gel mide
were
0.5 pg/ml ethidium bromide. Gels were photographed on a shortwave WVilluminator with Kodak Contrast Process Pan Film 4155 through a sandwich of Wratten No. 9 and No. 25 filters.
The exposure time
were determined by measuring distances
on
was
4-8 min.
Mobilities
photographs and assuming that the
The reproducibility rate of migration was constant during electrophoresis. of mobility values was examined in a few cases. These values showed a
standard deviation of ±7% or less. from individual experiments.
Values reported in the Figures and Tables
are
RESULTS
Agarose Concentration and Electrophoresis Voltage The effects of agarose concentration and electrophoresis voltage on
656
Nucleic Acids Research the separation of DNAs were examined using T4 DNA, X DNA and the restriction endonuclease-generated fragments of X DNA. Figure 1 and Table 1 show the
effect of varying the agarose concentration (at constant voltage, lV/cm of gel length) on the absolute and relative mobilities of these DNAs. Although the absolute DNA mobility (mm/hr) increases as the agarose concentration decreases from 0.7% to 0.2%, the mobility of each DNA relative to that of the 14 million molecular weight DNA decreases (Figure 1). This results in a compaction of the relative mobility distribution for DNAs below this molecular weight. For T4 DNA, X DNA and the 14 million molecular weight DNA,
however, there is a moderate expansion of the relative mobility distribution. The expansion can be clearly seen as an increase in the X DNA/T4 DNA mobility ratio at lower agarose concentrations (Table 1). This differential effect, along with the general increase in absolute mobility, greatly decreases the I I e a
I
I
I
I
I
I
I
I
I
"
0
.C G) 0 7 '07 : 10 -
0
ou
0
X AX % a 0\
l0
A
d
a b
I 0
1l1III 0.5
11
1.0 2.0 1.5 Relative Mobility
e
-
III 2.5
3.0
3.5
Figure 1. Effect of agarose concentration on DNA electrophoretic mobility. The voltage gradient was lV/cm of gel length. The lines represent: (a) 0.2% agarose without ethidium bromide, (b) 0.2% agarose, (c) 0.3% agarose, (d) 0.4% agarose and (e) 0.5% agarose. Mobilities are normalized to the mobility of Eco RI X DNA fragment A which had the following value in each gel: (a) 5.2 mm/hr, (b) 3.9, (c) 3.3, (d) 1.4 and (e) 1.2. The dashed line is the linear extrapolation of the data for low molecular weight DNAs in experiment a. 657
Nucleic Acids Research time required to separate X and T4 DNAs (Table 1). Further reduction in electrophoresis time is obtained by omitting ethidium bromide from the 0.2%
gel; a 1.0 cm separation of T4 and X DNAs requires only 12 hr. Ethidium bromide was included in most of the gels in this work because this allows visualization of the DNA during electrophoresis and eliminates the need to soak the gel in ethidium bromide for 5-10 hr at the end of the electrophoresis. For preparative work gels can be run without ethidium agarose
bromide and sections of the gel subsequently stained to locate DNA in unstained parallel sections. Figure 2 and Table 2 show the effect of varying the electrophoresis voltage (V/cm of gel length) on the mobility of these same DNAs in 0.2% As the voltage decreases from 2.5V/cm to 0.02V/cm the absolute agarose. mobilities of all the DNAs decrease. There is an expansion of the entire relative mobility distribution because mobilities of larger DNA molecules decrease by
larger factor than those of smaller DNA molecules. Most important, plots of log M versus mobility at lower voltages exhibit a straight line up to 32 million molecular weight (Figure 2). Separation of DNAs at lower voltages, of course, is slower. At O.lV/cm a 1.0 cm separation of T4 and X DNAs would require 92 hr in the presence of ethidium bromide (Table 2). Bacteriophage G DNA (500 Million Daltons) a
Electrophoretic separation of high molecular weight DNAs was studied further using DNA from bacteriophage G. This large virus has been reported
Table 1: Effect of agarose concentration on the electrophoretic separation of X and T4 DNAs. Agarose concentration (%)
0.2a 0.2 0.3 0.4 0.5 0.7
Mobility of T4 DNA
Mobility Ratio
Calculated time for one cm separation of X and T4 (hr)
(mm/hr)
X/T4
3.2 2.5 2.2
1.25
12
1.20
20
1.18 1.19 1.14 1.04
25
0.92 0.96 0.53
57 74 470
Data for the 0.2% to 0.5% agarose gels is taken from the experiments used to make Figure 1. Voltage for all gels was lV/cm. a
without ethidium bromide.
658
Nucleic Acids Research to contain a DNA molecule with a molecular mass and sequence complexity of approximately 500 million daltons (4, 9). G DNA, isolated as described in MATERIALS AND METHODS, was analyzed by zone sedimentation. Results such as
those shown in Figure 3 indicate that the procedures employed for isolation and manipulation of phage G DNA yield a fairly homogenous preparation of molecules of about 500 x 106 daltons. Various conditions of electrophoresis were examined with phage G DNA included as a molecular weight standard. The results of electrophoresis under conditions which achieve separation of X, T4 and G DNAs in a short time are summarized in Table 3 and Figure 4. In 0.2% agarose gels run at lV/cm (experiment d) the T4 DNA/G DNA mobility ratio is 1.10; a 1.0 cm separation can be achieved in 34 hr of electrophoresis. However, mobility in the X DNA to G DNA molecular weight range is nonlinear with log M of the DNA (Figure 4d). Larger DNAs in this range, therefore, would be more
poorly separated under these conditions. I
I
a
I
I
I
Decreasing the agarose concentraX
I
I'
b c de A* .
*
lOB
0 .0 .o
.:
.,
Nucleic Acids Research
Separation of very large DNA molecules by gel electrophoresis Walton L. Fangman Department of Genetics, SK-50, University of Washington,Seattle, WA 98195, USA
Received 16 December 1977 ABSTRACT Very large DNA molecules were separated by electrophoresis in horizontal slab gels of dilute agarose. Conditions of electrophoresis were developed using intact DNA molecules from the bacterial viruses A, T4 and G. Their DNAs have molecular weights (M) of 32 million, 120 million, and 500 million, respectively. Several electrophoresis conditions were found which give sufficiently high mobilities and large mobility differences that these DNAs are separated in a short time. Electrophoresis in 0.1% agarose at 2.5 V/cm of gel length separates T4 and X DNAs by 2.0 cm, and G and T4 DNAs by 1.0 cm in only 10 hr. With some conditions DNA mobilities are directly proportional to log M for M values from 10 to 500 million. The procedures used will allow rapid molecular weight determination and separation of very large DNA molecules. INTRODUCTION
The DNA molecules of eukaryotic chromosomes, chloroplasts and large viruses have molecular weights of 100 million and greater. Such large DNA molecules cannot easily be separated on the basis of size. Separation by sedimentation velocity suffers from zone spreading which probably results
from convection, and from the necessity of centrifuging for long periods at low centrifuge speed. At high centrifuge speeds DNA molecules exceeding 100 million daltons exhibit a marked reduction in s (see citations in ref. 1). Gel electrophoresis, which has not been applied to very large DNAs, has been extremely useful for separating smaller DNA molecules (less than 10 million daltons). Duplex DNA molecules differing by a few percent in molecular weight can be resolved. A plot of log M versus electrophoretic mobility approximates a straight line with large molecules having a slower rate of migration than smaller ones. However, with the electrophoresis conditions usually employed DNA molecules with a mass above 10 million daltons migrate much faster than expected and are not well resolved. There have been a large number of publications on the electrophoretic behavior of small, less than 25 million dalton, DNA molecules (for citations
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
653
Nucleic Acids Research This paper presents conditions for electrophoretic separation of DNA molecules of 10 to 500 million daltons. The work is an extension of earlier observations of Henckes et al. (ref. 3). Large DNAs employed in developing the electrophoresis conditions were the intact molecules from see ref. 2).
bacterial viruses X, T4 and G. X and T4 DNAs have been well characterized and have masses of 32 million and 120 million daltons, respectively. Bacteriophage G DNA has been reported to have a mass of 500 million daltons (4). DNA was prepared from bacteriophage G particles by procedures which eliminate breakage by shear and used as a molecular weight standard.
MATERIALS AND METHODS Bacteriophage DNA. T4D and XCI857 viruses containing 14C or 3H labels were prepared by standard procedures. Bacteriophage G and its host Bacillus megatherium PGH were obtained from Prof. G. Donelli. The phage was cloned and subsequently titred with beef extract broth (BEB) plates and top agar at 30°. BEB medium contains per liter: 3 g beef extract (BBL), 5 g peptone (Difco), 5 g NaCl, 8 x 105 moles MgSO4, 2 x 10 moles -34 MnSO and 10 moles CaClV final pH 7.1-7.2. Plates contained 10 g agar/l and top agar 7.5 g agar/l. Cells for plating were grown in BEB liquid at
Phage G for DNA isolation was prepared by infecting exponential phase cells in TYEM medium at a culture optical density (660 nm) of 1.5 with 2 x 108 phage per ml of culture. TYEM medium contains per liter: 10 g tryptone (Difco), 5 g NaCl, 0.5 g yeast extract (Difco) and MgSO4, MnSO4, and CaCl2 as in BEB, final pH 7.4-7.5. With vigorous aeration the culture lysed in 3-5 hours. The lysed culture was shaken slowly with chloroform for a few min, then centrifuged at 4,000 x g for 20 min. The supernatant (1-2 x 10 phage/ml) was centrifuged at 27,000 x g for 30 min and the
30°.
pellet containing the phage suspended with cold .OlM Tris, .OlM MgSO4, pH 7.4 (TM) containing 1 mg/ml bovine serum albumin (BSA). This material was incubated with 100 pg/ml RNase I for 15 min at room temperature, then 0.5-1.0 ml (containing 1-5 x 1011 phage) was layered onto a cold 16 ml linear gradient of 10-35% sucrose in TM-BSA. After centrifugation in a Spinco SW27.1 rotor at 14,000 rpm for 30 min (5°), the phage were collected as part of a white diffuse band. DNA was labeled by adding 2.5 pc/ml 6- H-uracil at 0, 1 and 2 hours of infection. DNA was isolated from G particles by very slowly mixing 0.7 ml of phage suspension with 0.7 ml 10% sodium lauroyl sarcosinate in 0.2M EDTA, pH 8 in a Spinco SW50 polyallomer centrifuge tube. The tube was corked
654 t
Nucleic Acids Research and heated at 650 for 10 min.
The solution
underlaid with 3.5 ml
was
temperature-saturated CsCl in 0.2M EDTA, pH 8 and centrifuged in rotor at 30,000 rpm for 2-3 days (100).
Fractions
were
an
room
SW50L
collected through
the tube bottom with a 13G hooded needle (boiled in EDTA solution) at a flow rate of 0.10-0.15 ml/min. The DNA was dialyzed in a Collodion bag (S & S) or dialysis tubing held open at one end with a ring of plastic, against 10 mM Tris, 100 mM EDTA, pH 8 and stored at 5°. For sedimentation analysis
or
gel electrophoresis G DNA
was
kept intact by employing slow
transfers with 1.0-1.5 mm i.d. pipettes. T4 and X DNAs were prepared from virus particles by the same procedure. The initial preparations of X and T4 DNAs used in this work contained intact DNA molecules based on the ratio of their S values and the contour lengths of the DNAs observed by electronmicroscopy (5, 6). Subsequent preparations were characterized by electrophoresis in 0.2% agarose as reported here.
The molecular weight of G DNA
mentation with T4 DNA (Figure 3).
was
determined by cosedi-
H-labeled G DNA (0.20 pg) and
C-
1.0 mM EDTA, pH8, was labeled T4 DNA (0.05 pg) in 0.5 ml 1.0 mM Tris in 0.O1M Tris, sucrose layered on a 58 ml linear gradient of 15 to 30% 0.5M NaCl. The gradient was centrifuged in a 0.2M EDTA, pH8 pH8 Spinco SW25.2 rotor at 8,000 rpm for 3.5 days (5°). Molecular weights -
-
-
(ref. 7). calculated from the equation S1/S2 = (M1/M2) Specific fragments of X DNA were generated with the endonucleases Sal I (New England Biolabs) and Eco RI (supplied by Dr. Maynard Olson and Guy
were
Page). Sizes for X DNA and X DNA fragments were taken from data of Dr. Peter Philippsen (personal communication) obtained from electron microscopic contour length measurements. His kilobase pair values were converted to mass units using 660 daltons/base pair to obtain masses, in millions of daltons, of 32 for intact X DNA, 21 and 10 for the two major Sal I fragments (A and B, respectively), and 14, 4.9, 3.8, 3.6, 3.1 and 2.2 for the six Eco RI fragments (designated here as A, B, C, D, E, and F, respectively). T4 DNA was taken to have a mass of 120 million daltons (8).
Electrophoresis.
Horizontal agarose gels were formed on a glass plate
plastic container with a raised center section separating two buffer reservoirs and electrodes. Buffer was in direct contract with the ends of
in
a
the gel.
Gels were 13.5 cm wide, 10 to 25 cm long and about 1.0 cm thick.
Seakem LE agarose was used for all gels except 0.1% agarose gels in which Seakem HGT(P) agarose was used to provide greater mechanical stability. 655
Nucleic Acids Research Gels consisting of less than 0.4% agarose
1.5
cm
made by first pouring
a
2
agarose were mm
slab, then four sides
This "box" allowed dilute gels
wide.
poured into
to be
picked
a 1
up
"box" of 1% cm
thick and
and moved for
photography and other manipulations. The electrophoresis buffer contained 10.3g Tris, 5.5g boric acid and 0.93g disodium EDTA per liter. For some gels the buffer contained 4.4
g
Tris, 4.1
g
anhydrous monosodium phosphate,
0.37 g disodium EDTA per liter. The two buffers gave only small differences in mobilities. Unless stated otherwise, gels and buffers contained 0.5 Pg/ml ethidium bromide.
Sample wells
ing ten 0.75
mm
x
8.0
in contact with the 1%
teeth. agarose
were
made with
The wells
were
bottom layer.
a
Biorad teflon comb contain-
about 8
mm
deep and
Samples of 30 pl
were
were
not
intro-
a screw-type pipettor and 50 p1 disposable glass micropipettes (1 mm i.d.). Gels of 0.1 and 0.2% agarose were chilled to 50 to stiffen them before removing the comb and
duced into the wells by slow hand pipetting using
loading the wells. The 30 p4 sample was 1 mM Tris, pH 8, 10 mM EDTA, pH 8, 10% glycerol and 0.0015% bromphenol blue and contained 0.05 ig Sal I A DNA fragments, 0.10 vg Eco RI X DNA fragments, and about 0.025 hg each of X DNA, T4 DNA and G DNA. Three-fold lower and three-fold higher amounts of the intact virus DNAs did not result in altered mobilities although streaking toward the wells occurred at the higher concentrations. The mobility of
each DNA run alone was the same as in the mixture. After loading, the entire gel was covered with household plastic wrap and electrophoresis was carried out at room temperature (about 210) at constant voltage. The voltage gradient was measured with a Midland voltmeter using platinum leads at a 10 cm spacing. Gels run without ethidium brosubsequently stained by soaking overnight in buffer containing
inserted into the gel mide
were
0.5 pg/ml ethidium bromide. Gels were photographed on a shortwave WVilluminator with Kodak Contrast Process Pan Film 4155 through a sandwich of Wratten No. 9 and No. 25 filters.
The exposure time
were determined by measuring distances
on
was
4-8 min.
Mobilities
photographs and assuming that the
The reproducibility rate of migration was constant during electrophoresis. of mobility values was examined in a few cases. These values showed a
standard deviation of ±7% or less. from individual experiments.
Values reported in the Figures and Tables
are
RESULTS
Agarose Concentration and Electrophoresis Voltage The effects of agarose concentration and electrophoresis voltage on
656
Nucleic Acids Research the separation of DNAs were examined using T4 DNA, X DNA and the restriction endonuclease-generated fragments of X DNA. Figure 1 and Table 1 show the
effect of varying the agarose concentration (at constant voltage, lV/cm of gel length) on the absolute and relative mobilities of these DNAs. Although the absolute DNA mobility (mm/hr) increases as the agarose concentration decreases from 0.7% to 0.2%, the mobility of each DNA relative to that of the 14 million molecular weight DNA decreases (Figure 1). This results in a compaction of the relative mobility distribution for DNAs below this molecular weight. For T4 DNA, X DNA and the 14 million molecular weight DNA,
however, there is a moderate expansion of the relative mobility distribution. The expansion can be clearly seen as an increase in the X DNA/T4 DNA mobility ratio at lower agarose concentrations (Table 1). This differential effect, along with the general increase in absolute mobility, greatly decreases the I I e a
I
I
I
I
I
I
I
I
I
"
0
.C G) 0 7 '07 : 10 -
0
ou
0
X AX % a 0\
l0
A
d
a b
I 0
1l1III 0.5
11
1.0 2.0 1.5 Relative Mobility
e
-
III 2.5
3.0
3.5
Figure 1. Effect of agarose concentration on DNA electrophoretic mobility. The voltage gradient was lV/cm of gel length. The lines represent: (a) 0.2% agarose without ethidium bromide, (b) 0.2% agarose, (c) 0.3% agarose, (d) 0.4% agarose and (e) 0.5% agarose. Mobilities are normalized to the mobility of Eco RI X DNA fragment A which had the following value in each gel: (a) 5.2 mm/hr, (b) 3.9, (c) 3.3, (d) 1.4 and (e) 1.2. The dashed line is the linear extrapolation of the data for low molecular weight DNAs in experiment a. 657
Nucleic Acids Research time required to separate X and T4 DNAs (Table 1). Further reduction in electrophoresis time is obtained by omitting ethidium bromide from the 0.2%
gel; a 1.0 cm separation of T4 and X DNAs requires only 12 hr. Ethidium bromide was included in most of the gels in this work because this allows visualization of the DNA during electrophoresis and eliminates the need to soak the gel in ethidium bromide for 5-10 hr at the end of the electrophoresis. For preparative work gels can be run without ethidium agarose
bromide and sections of the gel subsequently stained to locate DNA in unstained parallel sections. Figure 2 and Table 2 show the effect of varying the electrophoresis voltage (V/cm of gel length) on the mobility of these same DNAs in 0.2% As the voltage decreases from 2.5V/cm to 0.02V/cm the absolute agarose. mobilities of all the DNAs decrease. There is an expansion of the entire relative mobility distribution because mobilities of larger DNA molecules decrease by
larger factor than those of smaller DNA molecules. Most important, plots of log M versus mobility at lower voltages exhibit a straight line up to 32 million molecular weight (Figure 2). Separation of DNAs at lower voltages, of course, is slower. At O.lV/cm a 1.0 cm separation of T4 and X DNAs would require 92 hr in the presence of ethidium bromide (Table 2). Bacteriophage G DNA (500 Million Daltons) a
Electrophoretic separation of high molecular weight DNAs was studied further using DNA from bacteriophage G. This large virus has been reported
Table 1: Effect of agarose concentration on the electrophoretic separation of X and T4 DNAs. Agarose concentration (%)
0.2a 0.2 0.3 0.4 0.5 0.7
Mobility of T4 DNA
Mobility Ratio
Calculated time for one cm separation of X and T4 (hr)
(mm/hr)
X/T4
3.2 2.5 2.2
1.25
12
1.20
20
1.18 1.19 1.14 1.04
25
0.92 0.96 0.53
57 74 470
Data for the 0.2% to 0.5% agarose gels is taken from the experiments used to make Figure 1. Voltage for all gels was lV/cm. a
without ethidium bromide.
658
Nucleic Acids Research to contain a DNA molecule with a molecular mass and sequence complexity of approximately 500 million daltons (4, 9). G DNA, isolated as described in MATERIALS AND METHODS, was analyzed by zone sedimentation. Results such as
those shown in Figure 3 indicate that the procedures employed for isolation and manipulation of phage G DNA yield a fairly homogenous preparation of molecules of about 500 x 106 daltons. Various conditions of electrophoresis were examined with phage G DNA included as a molecular weight standard. The results of electrophoresis under conditions which achieve separation of X, T4 and G DNAs in a short time are summarized in Table 3 and Figure 4. In 0.2% agarose gels run at lV/cm (experiment d) the T4 DNA/G DNA mobility ratio is 1.10; a 1.0 cm separation can be achieved in 34 hr of electrophoresis. However, mobility in the X DNA to G DNA molecular weight range is nonlinear with log M of the DNA (Figure 4d). Larger DNAs in this range, therefore, would be more
poorly separated under these conditions. I
I
a
I
I
I
Decreasing the agarose concentraX
I
I'
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