Product Name:

Cat. #DS615
Box 1 (-20℃)

Box 2 (RT)DS625
Box 1 (-20℃)

Box 2 (RT)

日本語データシート

Long ssDNA Preparation Kit

Product
Long ssDNA Preparation Kit for 1.5 kb
pLSODN-1
pLSODN-2D
Denaturing Gel-Loading Buffer
Nicked pLSODN-1 (1.5 kb Fragment)
Long ssDNA Gel Extraction Kit for 3kb (#DS640)
Long ssDNA Preparation Kit for 3.0 kb
pLSODN-3
pLSODN-4D
Denaturing Gel-Loading Buffer
Nicked pLSODN-3 (3 kb Fragment)
Long ssDNA Gel Extraction Kit for 3kb (#DS640)

Size
10 μg
10 μg
1 ml
100 μl25 preps(0.5 μg/μl)(0.5 μg/μl)

10 μg
10 μg
1 ml
100 μl25 preps(0.5 μg/μl )(0.5 μg/μl )(0.1μg/μl, 50 loadings)(0.1μg/μl, 50 loadings)

Storage Conditions:Box 1 should be stored at -20℃.
Box 2 should be stored at 15-25℃, stable for up to 24 months from the date of receipt.

Introduction:

Single-stranded DNA (ssDNA) is widely used for molecular biology andbiotechnology applications. Several methods were developed to generate ssDNA from double-stranded
DNA (dsDNA), including asymmetric PCR, lambda exonuclease digestion, separation with denaturing-urea
PAGE or separation using streptavidin-coated magnetic beads (ref. 1, 2 and 3).
The Long ssDNA Preparation Kits give a simple and easy method for generation of a long ssDNA(pLSODN-1 and -2 for ssDNA fragments within 1,500 bases and pLSODN-3 and -4 for ssDNA fragments
within 3,000 bases). A long ssDNA prepared by this method has defined sequence and length because it
does not include inside mutation and terminal deletion caused by PCR, exonuclease side reaction,
non-high-fidelity reverse transcriptase reaction or non-high-fidelity synthetic oligonucleotides.
The procedure of this method is almost the same as the method to obtain dsDNA fragments. The DNA ofinterest is cloned into a plasmid. The resulting plasmid harboring the DNA is digested with a pair of two
nicking endonucleases or a combination of a nicking endonuclease and a restriction enzyme. The nicked
plasmid is denatured by mixing with Denaturing Gel-Loading Buffer and then subjected to agarose gelelectrophoresis. The band corresponding to a long ssDNA is excised and extracted with the Long ssDNA
Gel Extraction Kit.
The Long ssDNA Gel Extraction Kit is highly optimized for long ssDNA purification. It gives high yieldand high purity of a long ssDNA.

Additional Materials Needed:
To prepare a long ssDNA, the following materials are required.
• Nicking Endonuclease (Nt.BspQI, Nb.BsrDI, Nb.BtsI, Nb.BsmI or Nb.BbvCI)• Agarose for gel electrophoresis (e.g. Seakem GTGTM Agarose)• Ethidium bromide, SYBR® Green, crystal violet or any other DNA staining reagent• Ethanol and isopropanol.
• Heat block for 50-70℃ incubation for gel extraction.

Seakem® GTGTM Agarose and SYBR® Green are trademarks of FMC Corporation and Molecular Probes, Inc., respectively.

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KIT COMPONENT
1) Plasmids
These are plasmid maps of pLSODNs. Red boxes show the region containing nicking endonucleases.

2) Denaturing Gel-Loading Buffer
The Denaturing Gel-Loading Buffer is a gel-loading buffer to obtain a long ssDNA of interest using
agarose gel electrophoresis. The Denaturing Gel-Loading Buffer enables denaturing of the nicked pLSODN
plasmid harboring the DNA of interest. A mixture of the nicked plasmid and the Denaturing Gel-Loading
Buffer can be subjected to agarose gel electrophoresis. During electrophoresis, DNAs derived from thenicked plasmid keep single-strand status. To use, three volumes of the Denaturing Gel-Loading Buffer are
added to the nicked plasmid, then heated and chilled to load into wells of agarose gel.

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3) Nicked pLSODN-1 (1.5 kb Fragment) or Nicked pLSODN-3 (3 kb Fragment) *1
The Nicked pLSODN-1 (1.5 kb Fragment) in DS615 or Nicked pLSODN-3 (3 kb Fragment) in DS625 canbe used for molecular weight marker of long ssDNAs on analytical agarose gel electrophoresis.*2 Although
the nicked plasmids give only three bands, they are useful to monitor experiments and to identify a long
ssDNA of interest as the plasmid backbone is the same as the plasmids in the kit.*4
1

2

Figure 1. Electrophoresis profile of Nicked pLSODN-1 (1.5 kb Fragment)
and Nicked pLSODN-3 (3 kb Fragment) *1, *2, *3
1.2% agarose gel in 1x TAE at 100 V.
Lane 1: Nicked pLSODN-1 (1.5 kb Fragment)
Lane 2: Nicked pLSODN-3 (3 kb Fragment)
Both Nicked plasmids were denatured.The gel was stained with ethidium bromide under UV light.

*1

The pLSODN-1 (1.5 kb Fragment) was constructed by inserting a 1.5 kb fragment of DNA between the Nt.BspQI
and the Nb.BsrDI sites of pLSODN-1. The pLSODN-3 (3 kb Fragment) was also constructed in a similar way. The
double-nicked plasmids were obtained by double digestion with Nt.BspQI and Nb.BsrDI. The double-nicked
pLSODN-1 (1.5 kb Fragment) gives three linear ssDNAs (1,500 bases, 3,251 bases and 4,751 bases) and one
circular ssDNA (4,751 bases). Similarly, the double-nicked pLSODN-3 (3 kb Fragment) gives three linear ssDNAs(3,000 bases, 6,487 bases and 9,487 bases) and one circular ssDNA (9,487 bases).
*2 Both markers are supplied at a concentration of 0.1 μg/μl in 75% Denaturating Gel-Loading buffer. It isready-to-use. To use, heat and chill before loading into wells of agarose gel.
Although ssDNAs give relatively weaker bands than dsDNAs on ethidium bromide staining, 150 ng – 300 ng of
nicked plasmid is enough for analytical agarose gel electrophoresis.
*3 Two linear ssDNAs (4,751 bases and 9,487 bases) are derived from randomly broken circular ssDNAs. They
originate in an open circular form of plasmid DNA. The band of 4,751-base linear ssDNA often overlaps with theband of 4,751-base circular ssDNA on agarose gel electrophoresis.
*4 The Nicked pLSODN-1 (1.5 kb Fragment) or Nicked pLSODN-3 (3 kb Fragment) is very convenient in analyticalagarose gel electrophoresis. But if you need a highly purified long ssDNA without any impurities, we do notrecommend their use in preparative electrophoresis.

4) Long ssDNA Gel Extraction Kit for 3kb(# DS640)

Kit Components: 25 preps
Components
Contents
Crystal Violet Solution
Crystal Violet 4mg/ml (2,500 x).
Gel-Dissolving Buffer
It contains guanidine thiocyanate as chaotropic salts.
Wash Buffer 1
Tris-base buffer(Add 45 ml of 100 % ethanol to the Wash Buffer before use.)
Wash Buffer 2
Tris-base buffer(Add 45 ml of 100 % ethanol to the Wash Buffer before use.)
Elution Buffer
10 mM Tris-HCl buffer, pH 8.0.
Spin Column
Collection Tube
• If precipitated material has formed in Gel-Dissolving Buffer, heat to dissolve at 37℃。

Size
5 ml
45 ml
8 ml
11 ml
5 ml
25 pieces
25 pieces

The Long ssDNA Gel Extraction Kit is a highly-specialized kit for long ssDNA purification from agarosegel. The spin column, buffer formulations and purification protocol are optimized for high recovery yields

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and high purity of long ssDNA. Long ssDNA purified with the kit is high-quality, with low mechanicaldegradation and without ultraviolet light-induced damage from the purification process. The purified long
ssDNA is suitable for molecular biology and biotechnology applications.
The Long ssDNA Preparation Kit can be universally used for purification and clean-up of long ssDNA.

Features and Specifications:• Optimized for long ssDNA.
• High recovery yield (typically 75-90%).
• High purity.
• Low mechanical degradation.
• No UV light-induced DNA damage.
• Direct monitoring of the migration of long ssDNA blue bands in the gel during electrophoresis.
• Excision of long ssDNA under ambient light.
• Guanidine thiocyanate is used as chaotropic salts. NaI is not used.*1• ssDNA size for excision: 500 - 3,000 bases. *2• Binding capacity on a Spin Column for ssDNA binding is up to 5μg.
• Elution volume: ≥ 15 μl.
*1*2Residual NaI may be difficult to remove, and reduces the efficiency of downstream enzymatic reactions .
200 bases of long ssDNA also can be extracted using the kit, but the recovery yield is not so high (about
40-45%).

Caution!

Wear gloves and protective clothing while handling the Crystal Violet.

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Experimental Outline
The procedure to obtain a long ssDNA using this method is almost the same as that of a conventional
method to obtain dsDNA fragment. First, the DNA fragment of interest is cloned into the multiple cloningsite of pLSODN plasmid using at least one nicking endonucleases site. The resulting pLSODN plasmid
harboring the DNA fragment is digested with a pair of bottom and top-strand nicking endonucleases or a
combination of a nicking endonuclease and a restriction enzyme. The nicked plasmid is denatured and thensubjected to agarose gel electrophoresis. After electrophoresis, the gel is stained with DNA staining reagent.
The band corresponding to a long ssDNA is excised and extracted.
The table below describes the major steps necessary to obtain a long ssDNA of interest.
Step
STEP 1
STEP 2
STEP 3
STEP 4
STEP 5
STEP 6

ActionCloning of the DNA of your interest into pLSODN plasmid
Digestion of plasmid harboring the DNA
Desalting with ethanol precipitation
Analytical agarose gel electrophoresis
Preparative agarose gel electrophoresis
Gel extraction

Page
5-6
7
7
8
9-10
10-12

PRODUCT USAGESTEP 1: Cloning of the DNA of your interest into pLSODN plasmid
The sequences below are the multiple cloning sites of pLSODNs. To obtain a long ssDNA, it is necessary
to clone the DNA fragment of interest between appropriate nuclease cleavage sites in the multiple cloning
sites. Figure 2 shows a schematic representation of the possible choices of cloning sites to generate a long
ssDNA. There are three recommended methods:1.

Two nicking endonuclease sites (a bottom-strand nicking endonuclease and a top-strand nicking
endonuclease).

2.

An Nt.BspQI site and a restriction enzyme site (a top-strand nicking endonuclease and a restriction
enzyme generating 5’ overhang).

3.

An Nb type nicking endonuclease site and a restriction enzyme site (a bottom-strand nicking
endonuclease and a restriction enzyme generating 3’ overhang) or BbsI.

At least one nicking endonuclease site must be used for cloning to give three ssDNA fragments: a long
ssDNA (a), a linear single-stranded vector DNA (b) and a circular or linear single-stranded whole plasmid
DNA (c, d). Cloning sites should be chosen with consideration of nicking endonuclease sites and restriction
enzyme sites in the DNA fragment of interest and terminal sequences of the resulting long ssDNA. If there
are no appropriate cloning sites in the DNA fragment of interest because of the existence of some nicking
endonuclease sites or restriction enzyme sites in it, some of these sites in the DNA fragment may need to bechanged by site-directed mutagenesis not to affect the function of a long ssDNA, for example silent
mutation.
* If you want a highly purified ssDNA, the quality of the pLSODN plasmid harboring the DNA of interest isimportant. Amounts of host RNA, host genomic DNA, degradated plasmid DNA and open circular plasmid
DNA should be minimized.

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The multiple cloning sites of pLSODN-1 or pLSODN-3
M13 Rv primer
GTG GAA TTG TGA GCG GAT AAC AAT TTC ACA CAG GAA ACA GCT
LacZ
NotI
XbaI
SalI
BamHI
EcoRI
XhoI
NcoI
BglII
ATG ACC ATG ATT ACG CCA GCG GCC GCT CTA GAG TCG ACG GAT CCG AAT TCC TCG AGC CAT GGA GATMet Thr Met Ile Thr Pro Ala Ala Ala Leu Glu Ser Thr Asp Pro Asn Ser Ser Ser His Gly Asp
1
5
10
15
20
SpeI
HindIII Nb.BbvCI Nb.BsmI Nb.BtsI Nb.BsrDI SmaIEcoRV Nt.BspQIPstI
CTA CTA GTA AGC TTC CTC AGC GAA TGC GCA GTG GCA ATG CCC GGG ATG ATA TCG AAG AGC CTG CAG
Leu Leu Val Ser Phe Leu Ser Glu Cys Ala Val Ala Met Pro Gly Met Ile Ser Lys Ser Leu Gln
25
30
35
40
SphI
KpnI
SacI
ApaI
BmtI
NsiI
BbsI
M13 Fw primer
GCA TGC GGT ACC GAG CTC GGG CCC GCT AGC ATA TGC ATG TCT TCA CTG GCC GTC GTT TTA CAA CGT
Ala Cys Gly Thr Glu Leu Gly Pro Ala Ser Ile Cys Met Ser Ser Leu Ala Val Val Leu Gln Arg
45
50
55
60
65

The multiple cloning sites of pLSODN-2D or pLSODN-4D
M13 Rv primer
GTG GAA TTG TGA GCG GAT AAC AAT TTC ACA CAG GAA ACA GCT
LacZ
BbsI
NsiI
BmtI
ApaI
SacI
KpnI
SphI
PstI
ATG ACC ATG ATT ACG CCG AAG ACA TGC ATA GCT AGC GGG CCC GAG CTC GGT ACC GCA TGC CTG CAG
Met Thr Met Ile Thr Pro Lys Thr Cys Ile Ala Ser Gly Pro Glu Leu Gly Thr Ala Cys Leu Gln
1
5
10
15
20
Nt.BspQI
EcoRV
SmaI Nb.BsrDI Nb.BtsI Nb.BsmI Nb.BbvCI HidIII
SpeI
BglII
GCT CTT CGA TAT CAT CCC GGG CAT TGC CAC TGC GCA TTC GCT GAG GAA GCT TCA CTA GTA GAT CTC
Ala Leu Arg Tyr His Pro Gly His Cys His Cys Ala Phe Ala Glu Glu Ala Ser Leu Val Asp Leu
25
30
35
40
NcoI
XhoI
EcoRI BamHI
SalI
XbaI
NotI
M13 Fw primer
CAT GGC TCG AGG AAT TCG GAT CCG TCG ACT CTA GAG CGG CCG CGA CTG GCC GTC GTT TTA CAA CGT
His Gly Ser Arg Asn Ser Asp Pro Ser Thr Leu Glu Arg Pro Arg Leu Ala Val Val Leu Gln Arg
45
50
55
60
65
* Rectangular boxes show the nicking endonuclease sites. Downward and upward arrows show sense-strand nicking position andantisense-strand nicking position, respectively

Figure 2. A schematic representation of possible pairs of cloning sites to generate a long ssDNA③ An Nb type nicking endonucleases site and a restriction enzyme site
② An Nt.BspQI site and a restriction enzymesite① Two nicking endonucleases sites

Multiple Cloning Sites
HindIII
SpeI
BglII
NcoI
XhoII
EcoRI
BamHI
SalI
XbaI
NotI

Nb.BsrDI
Nb.BtsI
Nb.BsmI
Nb.BbvCI

Nt.BspQI

A

PstI
SphI
KpnI
SacI
ApaI
BmtI
NsiI

A

A

1
○

2
○

BbsI

3
○

D

C

D

B

B

B

At least one nicking endonuclease site must be used.

A: a long single-stranded DNA, B: a linear single-stranded vector DNA, C: a circular single-stranded whole plasmid DNA, D: a linear
single-stranded whole plasmid DNA

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STEP 2: Digestion of plasmid harboring the DNA
The plasmid harboring a DNA fragment of interest is digested with a pair of bottom and top-strand
nicking endonucleases or a combination of a nicking endonuclease and a restriction enzyme according toprotocols of enzyme manufacturers. One example of these procedures is shown below.
A 1.5 kb DNA fragment of interest was cloned between the Nt.BspQI and the Nb.BsrDI sites of pLSODN-1, theresulting plasmids were designated as pLSODN-1(1.5 kb Fragment). Similarly, pLSODN-2D (1.5 kb Fragment),
pLSODN-3 (3 kb Fragment) and pLSODN-4D (3 kb Fragment) were also constructed.
To generate a double-nicked plasmid, pLSODN-1(1.5 kb Fragment) was digested with Nt.BspQI and Nb.BsrDI as
follows. The following 50 μl reaction mixture was set up.
Component
plasmid
100 μg10x 3.1 NEBuffer
Nt.BspQI (10 unit/μl)
Nb.BsrDI (10 unit/μl)
dH2O

Volumevariable
5 μl
5 μl(50 unit)
5 μl(50 unit)
variable

Total volume
50 μl
The reaction mixture was incubated at 50℃ for 60 min and then incubated at 60℃ for 60 min.

STEP 3: Desalting with ethanol precipitation
After digestion, the nicked plasmid solution must be desalted with ethanol precipitation. Desalting of
nicked plasmid solution is very important for agarose gel electrophoresis of ssDNAs because residual
salt and Mg2+ prevent complete denaturation of the nicked plasmids. Imcomplete denaturation of the nicked
plasmid appears as a poor band of a long ssDNA on gel electrophoresis, resulting in low yield of the long
ssDNA.
The double-nicked plasmid shoud be desalted with ethanol precipitation as follows.
1. Add 1/10 volume of Sodium Acetate (3 M, pH 5.2) or NaCl (1M) to the nicked plasmid solution.*1, *2
2. Add 2.5 volumes ethanol or 0.7 volume isopropanol and mix well.
3. Incubate at room temperature or on ice for 10 min.
4. Spin at maximum speed for 10 min at 4℃- room temperature. Discard supernatant.
5. Spin again for 1 min. Discard residual supernatant completely with pipette tip.*3
6. Add 70% ethanol and vortex vigorously.*4
7. Spin for 5 min. Discard supernatant.
8. Spin again for 1min. Discard residual supernatant completely with pipette tip.
9. Repeat rinse with 70% ethanol (Step 8-12). *5
10. Dry the pellet.
11. Dissolve pellet in TE or dH2O.*6*1

Before ethnol precipitaton, denaturation of nicking enzyme or restriction enzyme with heat or phenol extraction
might be needed. In the case of Nt.BspQI and Nb.BsrDI, denaturation was not needed.
*2 Before addition of salt, NaCl concentration in reaction buffer should be checked. (e.g. already 100 mM NaCl in 1x
3.1 NEBuffer).
*3 Completely remove supernatant in order to desalt sufficiently.
*4 The pellet might be swimming in the 70% EtOH but it is OK. Don’t be afraid of the pellet dissolving, be afraid of
any salt remaining. Also, as desalination progresses, the pellet becomes viscous.
*5 Rinse twice with 70% ethanol in order to desalt sufficiently.
*6 The final concentration of nicked plasmid might be 1-3 μg/μl. It may be easy to handle.

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STEP 4: Analytical agarose gel electrophoresis
The nicked plasmid obtained in the above procedure is analyzed with agarose gel electrophoresis.
1. Prepare loading samples by mixing the nicked plasmid and 3 volumes of the Denaturing Gel-Loading
Buffer.*1, *2, *3
2. Heat the mixture at 70°C for 5 min, then chill on ice for 1 min. Subject the mixture to 1.2% agarose
gel electrophoresis with 1 x TAE. Run at 100 V constant voltage.*4, *5
3. After electrophoresis, immerse the gel in a solution containing 0.5 μg/ml of ethidium bromide forapproximately 20 min. Then examine the gel on a UV illuminator (figure 3).
1

2

3

4

5

6

7

8

9

10

bp
10,000
7,000
5,000
4,000
3,000
2,500
2,000
1,500
1,000
700
500, 525
300

Figure 3. Analytical agarose gel electrophoresis of denatured and double-nicked plasmids.
Lane 1:
Lane 2:
Lane 3:
Lane 4:Lane 5:
Lane 6:
Lane 7:
Lane 8:Lane 9:Lane10:pLSODN-1 (1.5 kb Fragment)
Nicked pLSODN-1 (1.5 kb Fragment)
pLSODN-2D (1.5 kb Fragment)
Nicked pLSODN-2D (1.5 kb Fragment)
pLSODN-3 (3 kb Fragment)
Nicked pLSODN-3 (3 kb Fragment)
pLSODN-4D (3 kb Fragment)
Nicked pLSODN-4D (3 kb Fragment)
DynaMarker® DNA High D, denatured*6
DynaMarker® DNA High D, non-denatured

112.5 ng
250 ng112.5 ng
250 ng112.5 ng
250 ng112.5 ng
250 ng
5 μl (loading volume 20 μl)
5 μl

1.2% agarose gel in 1x TAE. Nicked plasmids in Lane 2, 4, 6 and 8 and DynaMarker® DNA High D in Lane 10 weredenatured. Plasmids in Lane 1, 3, 5 and 7 and DynaMarker® DNA High D in Lane 9 were not denatured. The gel was stainedwith ethidium bromide under UV light. The blue, orange and red arrows show long ssDNAs, linear single-stranded vector
DNAs and circular single-stranded whole plasmid DNAs, respectively. The yellow arrow shows randomly broken circularsingle-stranded whole plasmid DNAs. They originate in an open circular form of plasmid DNA.

*1

*2
*3

*4
*5
*6

Desalting of the nicked plasmid solution in advance is important. If the nicked plasmid solution contains
excessive salt and Mg+2, it cannot be completely denatured even by mixing with the Denaturing Gel-Loading
Buffer, resulting in a weak band of long ssDNA of interest (see “STEP 3: Desalting with ethanol precipitation”
at page 7).
Although ssDNAs give relatively weaker bands than dsDNAs on ethidium bromide staining, 150 ng – 300 ng
of nicked plasmid is enough for analytical agarose gel electrophoresis.
After mixing with the Denaturing Gel-Loading Buffer, the concentration of the nicked plasmid in theloading sample should be under 0.5 μg/μl. If it exceeds 0.5 μg/μl, the band of a long ssDNA is weakened asa result of incomplete denaturation. It results in a low recovery yield of the long ssDNA. The acceptablemaximum concentration of a nicked plasmid may vary depending on the melting temperature of the long
ssDNA. It could be checked by carrying out analytical agarose gel electrophoresis by loading several
concentrations of the nicked plasmid.
The heating and chilling procedure of samples should be carried out just before loading.
The composition of 1×TAE buffer is 40 mM Tris, 20 mM acetic acid and 1 mM EDTA (ref. 6).
Commercially available DNA molecular weight markers sometimes could not give clear bands after mixing
with Denaturing Gel-Loading Buffer. It may be caused by different migration of complementary strands due totheir secondary structure or incomplete denaturation.

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STEP 5: Preparative agarose gel electrophoresis
The nicked plasmid is subject to preparative agarose gel electrophoresis. The procedure is similar to thatof analytical gel electrophoresis.
ⅰ) Preparation of 1.2% agarose gel containing Crystal Violet
An example of this is given for making 100 ml of a 1.2% agarose gel.
1.

Add 1.2 g of agarose powder to 100 ml of 1×TAE buffer and mix them.

2.

Dissolve agarose in the microwave.

3.

Add 40μl of Crystal Violet Solution to the agarose (100 ml) and mix them by swirling.

4.

Pour the agarose into a mold and set a comb.* Allow the gel to harden.

*

It is recommended to make thick gel with deep wells so as not to cause the overflow of a loaded sample in a well,resulting in contamination.

ⅱ) Preparative agarose gel electrophoresis and excision
A simple way is shown in the following example.
1.

Pre-chill 1xTAE running buffer containing Crystal Violet (add 40μl Crystal Violet /100ml 1xTAE) on
ice or in a refrigerator. *1
Put the horizontal gel electrophoresis apparatus on ice in an ice bucket in a draft chamber. *2, *3, *4

2.

3. Pour pre-chilled 1xTAE running buffer containing Crystal Violet into the electrophoresis apparatus. *5
4.

Put the agarose gel into the electrophoresis apparatus.

5. Prepare loading samples by mixing the long ssDNAs sample (e.g. the nicked plasmid) and 3 volumes of the
Denaturing Gel-Loading Buffer. *6, *7
6.

Heat the mixture at 70°C for 5 min, then chill on ice for 1 min. Subject the mixture to agarose gelelectrophoresis. *8, *9, *10

7.

Run the gel at 100 V at a low temperature (less than 20℃). *2, *3, *4

8.

Monitor the long ssDNA blue bands moving in the gel during electrophoresis (Figure 1). Stop theelectrophoresis after the bands are sufficiently resolved. *11, *12
Excise the long ssDNA band of interest from the agarose gel with a scalpel.*13

9.
10.

1

2
Figure 4. Agarose gel electrophoresis using the gel containing Crystal Violet.
1.2% agarose gel in 1x TAE, 5.5cm x 6.0 cm. The loading volume of each sample is 24 μl.
Lane 1: Nicked pLSODN-1 (1.5 kb Fragment) 12 μg
Lane 2: Nicked pLSODN-3 (3 kb Fragment)
12 μg
3.0 kb (2μg)
1.5 kb (2μg)*1*21xTAE with Crystal Violet (add 40 μl Crystal Violet /100ml TAE) should be used as running buffer.
For the safe use of Crystal Violet, electrophoresis with Crystal Violet should be carried out in a draft chamber.
Don’t do electrophoresis with Crystal Violet or ethidium bromide in a closed space (especially in a cold room).
These dyes diffuse into the air with the water vapor or bubbles generated by electrolysis during electrophoresis.

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*3

Electrophoresis at a low temperature is recommended (less than 20℃). It gives a better resolution and strong
Crystal Violet staining. If the temperature of gel and running buffer exceeds 25℃, Crystal Violet staining of long
ssDNA becomes weak. If so, the gel can be restained by gentle shaking in a dilute Crystal Violet solution (40 μl of
Crystal Violet Solution / 100 ml of water) for 0.5 to 1 hour after electrophoresis.
*4 To keep the gel temperature low during electrophoresis in a draft chamber, we recommend placing theelectrophoresis apparatus on ice in an ice bucket.
*5 At least, use a pre-chilled 1xTAE running buffer on ice to keep a low temperature.
*6 Desalting of the nicked plasmid solution in advance is important for agarose gel electrophoresis. If the nicked
plasmid solution contains excessive salt and Mg+2, it adversely affects denaturation, resulting in giving poor bands
and low recovery yields of the long ssDNA of interest. If your long ssDNA band is extremely weak, first you
should consider the existence of residual salt in the long ssDNAs sample (or the nicked plasmid) solution. We
recommend removing the salt in the long ssDNAs sample through ethanol precipitation (see “STEP 3: Desaltingwith ethanol precipitation” at page 7)..
*7 To get a highly purified long ssDNA, the long ssDNA of interest should be loaded greater than 1μg/band. At thesame time, the concentration of the nicked plasmid in the loading sample should be under 0.5 μg/μl after
mixing with the Denaturing Gel-Loading Buffer.
*8 The heating and cooling procedure of the long ssDNAs sample (e.g. the nicked plasmid) mixture should be carriedout just before loading into wells of the gel.
*9 Do not load a sample too much in a well because it may overflow and cause contamination. It is recommended tomake deep wells with thick gel.
*10 Long ssDNA bands moving in Crystal Violet-containing gel can be directly seen under ambient light in realtime during electrophoresis. The electrophoresis can be stopped as soon as the bands are sufficiently resolved.
*11 Because crystal violet (positively-charged molecule) migrates toward the cathode, the dye concentration of therunning buffer and the gel on the anode side gradually decreases. Check the dye concentration and if needed, add
dye into the running buffer on the anode side during electrophoresis (e.g. 25 μl of Crystal Violet Solution is added
to 250 ml running buffer on the anode side every 30 min at 100V constant voltage).
*12 The long ssDNA band can be visible as a blue band and can be easily excised from the agarose gel on the bench.
*13 Minimize the size of the gel slice by removing extra agarose.

DNA f
DNA f

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STEP 6: DNA extraction and purification
Before start:• Add 45 ml of 100 % ethanol to Wash Buffer 1 and Wash Buffer 2 bottles, respectively.
• Isopropanol is required in the step.
• All centrifugation steps should be carried out at 16,000×g (around 13K rpm in a conventional
microcentrifuge) at room temperature (20℃- 25℃). Centrifugation at lower temperature might affectlong ssDNA yield.
• This kit can also be used for long ssDNA clean-up from enzymatic reactions. For this purpose, add 3
volumes of Gel-Dissolving Buffer and 1 volume of isopropanol to the reaction, mix well, and proceedwith step 5 of the protocol.
1.

Transfer the gel slice to a 1.5 ml microcentrifuge tube and weight the gel slice.

2.

Add 3 volumes of Gel-Dissolving Buffer.

3.

Incubate the tube at 50℃, vortexing periodically until the gel slice is completely dissolved for 10-15 min. *1

4.

Add one gel volume of isopropanol to the dissolved gel and mix well.

5.

Insert a Spin Column into a Collection Tube.

6.

Load the sample to the Spin Column and centrifuge for 1 min. Discard the flow-through in the Collection
Tube with a 1ml pipette tip. *2, *3, *4

7.

Centrifuge the Spin Column again for 1 min. Remove the residual flow-through completely with a 10 μl or a
100 μl pipette tip. *5

8.

Add 500 μl of Wash Buffer 1 to the Spin Column and centrifuge for 1 min. Discard the flow-through in theCollection Tube with a 1ml pipette tip (1st Wash). *3, *4

9.

Repeat wash with Wash Buffer 1 (Step 8, 2nd Wash).

10. Add 500 μl of Wash Buffer 1 to the Spin Column, close the cap tightly and vortex for 5 seconds to wash thewhole inner wall of the Spin Column (3rd Wash).
11. Centrifuge for 1 min. Keep the flow-through in the Collection Tube and vortex for 5 seconds to wash boththe outer wall of the Spin Column and the inner wall of the Collection Tube.
12. Centrifuge for 1 min. Discard the flow-through in the Collection Tube using a 1ml pipette tip. *3, *4
13. Add 500 μl of Wash Buffer 2 to the Spin Column and centrifuge for 1 min. Discard the flow-through in theCollection Tube with 1ml pipette tip (4th Wash). *3, *4
14. Centrifuge the Spin Column again for 1 min to remove residual Wash Buffer 2 completely.
15. Transfer the Spin Column into a new microcentrifuge tube.
16. Add 15-40 μl of Elution Buffer onto the Spin Column and incubate it at 70℃ for 5 min. *6
17. Directly Centrifuge the Spin Column for 1 min to elute long ssDNA. *7, *8, *9

1

2

3

4

Figure 5. Agarose gel electrophoresis of purified long ssDNAs.
1.2% agarose gel in 1x TAE. 5.5cm x 6.0 cm.
Lane 1: Nicked pLSODN-1 (1.5 kb Fragment)
800 ngLane 2: a long ssDNA purified from pLSODN-1 (1.5 kb Fragment) 400 ng
Lane 3: Nicked pLSODN-3 (3 kb Fragment)
800 ng
3.0 kb (0.4μg) Lane 4: a long ssDNA purified from pLSODN-3 (3 kb Fragment) 400 ng
1.5 kb (0.4μg)

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Ver.1.4

*1
*2
*3
*4

*5
*6
*7

*8

*9

Dissolve the agarose completely. If not, it might decrease the recovery yield of long ssDNA and cause
contamination of agar or buffers.
If the volume of dissolved sample exceeds 500 μl, the loading of the sample onto the column should be performed
in multiple rounds not to exceed 500 μl.
Don’t flip the Collection Tube to discard the flow-through. It contaminates the edge and upper inner wall of theCollection Tube. Use a 1 ml pipette tip to discard the flow-through.
Be careful to ensure the column tip does not contact column flow-through. If possible, we recommend using a new
Collection Tube at several washing steps to avoid contamination. However, spare collection tubes are not includedin the kit. They can be purchased from many companies (e.g. Corning Axygen #MCT-200-NC).
Don’t wash the Spin Column with Gel-Dissolving Buffer only.
Typical elution volumes are 15 μl-40 μl. Water can be used to elute the long ssDNA. The average elution volume
is 38 μl from 40 μl Elution Buffer volume, and 13 μl from 15 μl.
The total yield of ssDNA recovery from a 12 μg of a nicked plasmid pLSODN-1(1.5 kb Fragment) or pLSODN-3(3 kb Fragment) was about 1.7 μg (100% recovery is 2 μg) using one column of the Long ssDNA Gel Extraction
Kit for 3 kb with 40 μl Elution Buffer.
Generally, samples eluted from silica based spin columns contain a small amount of silica matrix. If necessary, it
could be easily removed by spin down (e.g. 20,000×g for 10 min) or filtration using filter spin column (e.g.
Merck Ultrafree-MC GV 0.22 μm).
When quantifying the amount of lssDNA using a spectrophotometer, it is recommended to remove contaminatedsilica matrix by spinning down and collecting the supernatant before measurement. Presence of silica matrix cancause UV scattering, resulting in a uniform elevation of the baseline between 220nm to 320nm, and potentiallyleading to an overestimation of lssDNA quantity.

Troubleshooting Guide, FAQ
TROUBLEPOSSIBLE
CAUSE

The ssDNA band does notclearly separate during
electrophoresis.
Salt remaining
Low ssDNA yield

Is it possible to prepare
ssDNA smaller than
1.5 kb using Long ssDNA
preparation kit for 3.0kb(Cat. No. DS625)?

SOLUTION
Desalting of the nicked plasmid solution is very important.
ssDNA bands may not separate clearly if salt remains.
Wash thoroughly with 70% ethanol in ethanol precipitation.
Specifically, wash with 70% ethanol twice and vortex vigorously
during washing. After removing the supernatant bysedimentation or washing operation, centrifuge again to collect
the residual liquid on the inner wall surface and remove it
thoroughly with a micropipette.

In principle, it is also possible to prepare ssDNA smaller than 1.5 kb with this kit, Long
ssDNA Preparation Kit for 3kb (Cat. No. DS625).
However, "Long ssDNA Preparation Kit for 1.5kb (Cat. No. DS615)" is more suitable
for the preparation of ssDNA smaller than 1.5 kb. We recommend you to use #DS615. Inorder to obtain the same amount of ssDNA with #DS625 as compared with #DS615 alarger amount of plasmid is needed. Also, as the concentration of plasmid to apply toelectrophoresis gel is limited (about 0.5 μg/μl), the ssDNA concentration in the bandalso decreases. This may affect the purification degree and yield.

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BioDynamics Laboratory Inc.
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Ver.1.4

References
1. Wakimoto Y, Jiang J, Wakimoto H. (2014) Isolation of single-stranded DNA.
Curr Protoc Mol Biol. 1;107:2.15.1-9.
2. Avci-Adali M, Paul A, Wilhelm N, Ziemer G, Wendel HP. (2009) Upgrading SELEX technology byusing lambda exonuclease digestion for single-stranded DNA generation. Molecules. 24;15(1):1-11.
3. Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T. (2016) ssODN-mediatedknock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Commun. 20;7:10431.
4. Gu H, Breaker RR. (2013) Production of single-stranded DNAs by self-cleavage of rolling-circle
amplification products. Biotechniques. 54(6):337-43.
5. Rand KN. (1996) Crystal violet can be used to visualize DNA bands during gel electrophoresis and to
improve cloning efficiency. Elsevier Trends Journals Technical Tips Online. 1(1); 23–24.
6. Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY.

Related Products:
DS611 Denaturing Gel-Loading Buffer
DS612 Denaturing Gel-Loading Buffer
DS635 Long ssDNA Preparation Kit for 10 kb
DS650 Long ssDNA Gel Extraction Kit for 10 kb
DM122 DynaMarker® DNA High D
DS230 JetGiga Competent Cell (DH5α)

1 ml x 5
1 ml x 2(500 loadings)(200 loadings)

1 ml(100 loadings)
100 μl x 10

Purchaser Notification
The product shall be used by the purchaser for internal research purpose only. The products and plasmids
that are modified or harboring a DNA may not be transferred to third parties, resold, modified for resale, orused to manufacture commercial products or to provide a service to third parties without prior written
approval of Biodynamics laboratory Inc.

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BioDynamics Laboratory Inc.
www.biodynamics.co.jp

Ver.1.4