Long ssDNA Preparation Kit for 10kb

Cat. #
DS635

Product
Long ssDNA Preparation Kit for 10kb
pLSODN PCR Template
Denaturing Gel-Loading Buffer (DS611, DS612)
λ/HindIII DNA for long ssDNA preparation kit (DS670)
Long ssDNA Gel Extraction kit for 10kb (DS650)

日本語データシート

Size
20 μl
1 ml
200 μl25 preps(25 ng/μl)

Storage Conditions:pLSODN PCR template should be stored at -80℃.
Denaturing Gel-Loading Buffer should be stored under -20℃.
λ/HindIII DNA for long ssDNA preparation kit should be stored at -80℃.
Long ssDNA Gel Extraction kit for 10kb should be stored at 15-25℃, stable for up to 2 years from thedate of receipt.

Introduction:
3-10 kb long single-stranded DNA (ssDNA) can be prepared simply and easily with this kit. A long
ssDNA prepared by this kit has defined sequence and length. It does not include inside mutation andterminal deletion caused by PCR, exonuclease side reaction, non-high-fidelity reverse transcriptase
reaction or non-high-fidelity synthetic oligonucleotides.
In this method, at first the DNA fragment of interest is cloned into a plasmid. This plasmid isdigested with a pair of two nicking endonucleases or combination of nicking endonuclease andrestriction enzyme. The nicked plasmid is denatured by Denaturing Gel-Loading Buffer and subjected
to agarose gel electrophoresis. The band of long ssDNA is excised and extracted with Long ssDNA Gel
Extraction Kit for 10kb.

Additional Materials Needed:
To prepare a long ssDNA, the following materials are required.
• DNA polymerase for PCR suitable for long amplicons (PrimeSTAR GXL DNA Polymerase (Takara
Bio Inc.) is recommended)• DpnI• DNA extraction kit from agarose gel (e.g. Monarch DNA Gel Extraction Kit (New England Biolabs
Inc.))• DNA seamless assembly kit (NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs Inc)
is recommended).
• Competent cell which has high competency (e.g. DH5α).
• Plasmid purification kit (e.g. QIAGEN plasmid kit (QIAGEN N.V.))• Nicking Endonuclease (Nt.BspQI, Nb.BsrDI, Nb.BtsI, Nb.BsmI or Nb.BbvCI)• Agarose for gel electrophoresis (Seakem GTG Agarose (Lonza Rockland, Inc.) is recommended)
The sequence of pLSODN PCR Template:
You can obtain the sequences of pLSODN PCR template from our website.
(https://www.biodynamics.co.jp/products/ssdna/)
You will find the access ID to open the files inside the package.

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Kit Components
pLSODN PCR template
pLSODN PCR Template has pUC Ori and Ampicillin resistance gene.
The following Nicking Endonuclease sites and Restriction Enzyme sites are not present in pLSODN PCR
Template, and these enzymes can be used to prepare ssDNA.
Nicking Endonucleases available for ssDNA preparation:
Nt.BspQI, Nb.BbvCI, Nb.BsrDI, Nb.BtsI, Nb.BsmIRestriction Enzymes available for ssDNA preparation:
AatII, AbsI, AflII, AgeI, ApaI, AscI, AvrII, BamHI, BbsI, BbvCI, BglII, BmrI, BmtI, BsaI, BsgI,
BsiWI, BsmBI, BsmI, BspEI, BspQI, BsrDI, BsrGI, BssHII, BtgZI, BtsI, EagI, EcoRI, EcoRV, FseI,
HindIII, KasI, KpnI, MauBI, MfeI, MluI, MreI, NcoI, NgoMIV, NotI, NsiI, PciI, PstI, PvuII, SacI,
SalI, SbfI, SgrDI, SmaI, SpeI, SphI, TspMI, XbaI, XhoI

Denaturing Gel-Loading Buffer
The Denaturing Gel-Loading Buffer is a gel-loading buffer to obtain a long ssDNA using agarose gelelectrophoresis. The Denaturing Gel-Loading Buffer denature the nicked plasmid harboring the DNA ofinterest. A mixture of the nicked plasmid and Denaturing Gel-Loading Buffer is subjected to agarose
gel electrophoresis. During electrophoresis, DNAs derived from the nicked plasmid keep single-strand
status.
To use, three volumes of the Denaturing Gel-Loading Buffer are added to the nicked plasmid, thenheated and chilled to load into wells of agarose gel.
λ/HindIII DNA for long ssDNA preparation kit
The approximate size of ssDNA can be estimated by λ / HindIII DNA for long ssDNA preparation kit.
The mobility of ssDNA is more changeable depending on the sequence than dsDNA, therefore λ /HindIII DNA indicate the rough size.
To use, aliquot 20 μl into new tube, heat and chill before loading into wells of agarose gel. See figure 3
for fragment size.

Long ssDNA Gel Extraction Kit for 10kb (# DM650)
Kit Components: 25 reactions
Components
Contents or Comments
Size
Crystal Violet Solution
Crystal Violet 4mg/ml (2,500×).
5 ml
Gel-Dissolving Buffer
It contains guanidine thiocyanate.
45 ml
Tris-base buffer
Wash Buffer 1
8 ml(Add 45 ml of 100 % ethanol to the Wash Buffer before use.)
Tris-base buffer
Wash Buffer 2
11 ml(Add 45 ml of 100 % ethanol to the Wash Buffer before use.)
Elution Buffer
10 mM Tris-HCl buffer, pH 8.0.
5 ml
Spin Column
25 pieces• If precipitated material has formed in the Gel-Dissolving Buffer, heat to dissolve at 37℃。
The Long ssDNA Gel Extraction Kit is a highly-specialized kit for long ssDNA (lssDNA) purificationfrom agarose gel. The spin column, buffer formulations and purification protocol are optimized for highrecovery yields and high purity of lssDNA. LssDNA purified with the kit is high-quality, with low
mechanical degradation and without ultraviolet light-induced damage from the purification process. The
purified lssDNA is suitable for molecular biology and biotechnology applications.
The Long ssDNA Preparation Kit can be universally used for purification and clean-up of lssDNA.
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Features and Specifications of the Long ssDNA Gel Extraction Kit for 10kb (# DS650) :• Optimized for long ssDNA.
• High recovery yield (Typically 55-75%).
• 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.*• ssDNA size for excision: 3,000-20,000 bases.
• Binding capacity on a Spin Column for ssDNA binding is up to 10μg.
• Elution volume: ≥ 15 μl.
*

Residual NaI may be difficult to remove, and reduces the efficiency of downstream enzymatic reactions .

Caution!
Wear gloves and protecting clothing while handling the Crystal Violet.

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Experimental Outline
Using this kit, long ssDNA can be obtained by almost the same procedure as the conventional methodfor dsDNA preparation.
At first, the DNA fragment of interest is cloned into the PCR product of “pLSODN PCR Template” byseamless cloning. At cloning, a nicking endonuclease site or restriction enzyme site is put between theprimer and the insert (at least one nicking endonuclease site). This cloned plasmid is digested with a pairof nicking endonucleases or a combination of nicking endonuclease and restriction enzyme. The nicked
plasmid is denatured and then subjected to agarose gel electrophoresis. The band of long ssDNA is excisedand extracted.
The table below describes the procedure of long ssDNA preparation.
OutlineSTEP
1Design of Construct

PCR and purification ofpLSODN PCR Template

Cloning of insert
into pLSODN plasmid

Digestion of the plasmidwith nicking
endonucleaseand extractionof long ssDNA

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

ActionSelection of Nicking Endonuclease and Restriction
EnzymeDesign of PCR primer
PCR using pLSODN PCR template
Digestion with DpnI
Electrophoresis
Isopropanol precipitation
Electrophoresis and gel extraction
Preparation of insert
Ligation and transformation
Colony PCR
Plasmid purification
Confirmation of insert size by restriction enzyme
Sequencing of insert
Digestion of plasmid with nicking endonuclease
DesaltingAnalytical agarose gel electrophoresis
Preparative agarose gel electrophoresis
Extraction of long ssDNA

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5’ end of Insert
Recognition siteof enzyme

ForwardPrimerReversePrimer

3’ end of Insert
Recognition site of enzyme
Amp
PUC OripLSODNPCR template

PCR
InsertInsertRecognition siteof enzyme

pLSODN vector

Seamless Cloning

Recognition siteof enzyme

5’

Insert3’

ForwardPrimerReversePrimerAmp
PUC Ori

Constructed
Plasmid

Figure 1. The flow of plasmid construction

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Diagram of insertion site
”Nt” Nicking endonucleaseor
Restriction enzyme
generating 3’ overhang etc.

”Nb” Nicking endonucleaseor
Restriction enzyme
generating 5’ overhang etc.

3’5’3’ end ofpLSODNPCR Template

Nt.BspQI 5’ GCTCTTCN 3’
3’ CGAGAAGN 5’

Insert5’ NNCATTGC 3’ Nb.BsmI
3’ NNGTAACG 5’
5’ NNCACTGC 3’ Nb.BsrDI
3’ NNGTGACG 5’

5’ end ofpLSODNPCR Template

5’ NGCATTC 3’ Nb.BtsI
3’ NCGTAAG 5’
5’ GCTGAGG 3’ Nb.BbvCI
3’ CGACTCC 5’
PstI
etc.

5’ CTGCAG 3’
3’ GACGTC 5’

5’ AAGCTT 3’
3’ TTCGAA 5’

HindIII
etc.

Digestion
3 digestion patterns

A

A

A

D

C
B

B

Nt.BspQI anda restriction enzyme

Two nicking endonuclease

D
B

A restriction enzyme and“Nb” nicking endonuclease

At least one nicking endonuclease site must be used
A: a long single-stranded DNA of interest, B: a linear single-stranded vector DNA,
C: a circular single-stranded whole plasmid DNA, D: a linear single-stranded whole plasmid DNA

Figure 2. The possible pairs of recognition site to generate a long ssDNA

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DESIGN OF CONSTRUCT
Selection of nicking endonuclease and restriction enzyme, and design of PCR primer

STEP 1: Selection of Nicking Endonuclease and Restriction Enzyme
ssDNA can be prepared from the constructed plasmid using “two nicking enzymes” or “nicking
enzymes and restriction enzymes” that satisfy the following three conditions.
(1) The enzymes are in the list of below "Nicking enzymes and restriction enzymes available for
ssDNA excision".
(2) There is no recognition site of the enzymes inside your insert.
(3) The enzymes cleave the same strand of dsDNA.
If it is necessary to minimize extra bases at the end of ssDNA from the recognition site of enzymes,
consider the design shown in Figure 2 and below.
Nicking endonucleases and restriction enzyme available for ssDNA preparation
The site suitable for adding to 5’end of insert

The site suitable for adding to 3’end of insert ト

Nt.BspQI *1

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

AatII, ApaI, BbsI, BmrI, BmtI, BsaI, BsgI, BsmBI, BsmI,
BspQI, BsrDI, BtgZI, BtgZI, BtsI, FseI, KpnI, NsiI, PstI,
SacI, SbfI, SphI *2

AbsI, AflII, AgeI, AscI, AvrII, BamHI, BbvCI, BglII,
BsiWI, BspEI, BsrGI, BssHII, EagI, EcoRI, HindIII, KasI,
MauBI, MfeI, MluI, MreI, NcoI, NgoMIV, NotI, PciI, SalI,
SgrDI, SpeI, TspMI, XbaI, XhoI *2

*1
*2

*1

Nicking endonucleases are shown in red.
Besides these enzymes, EcoRV, PvuII and SmaI which generate blunt ends can also be used.

5’ GCTCTTCN 3’or
3’ CGAGAAGN 5’
Nt.BspQI

5’ CTGCAG 3’
3’ GACGTC 5’
PstI

5’ GCTGAGG 3’
3’ CGACTCC 5’
Nb.BbvCIor

5’ AAGCTT 3’
3’ TTCGAA 5’
HindIII"Nb." Nicking endonuclease or restriction enzymes
that leave 5' overhangs are suitable for attaching to the
3' side of insert (attach to forward primer of pLSODN
PCR template).
If the terminal sequence of the insert is shared withthe restriction enzyme site, the extra sequence will not
be added to the end of the prepared ssDNA. For
example, at Nb.BbvCI site, a single strand is cleaved
after “GC” indicated by the arrow as shown below.
Therefore, if the 3 'end of insert is “GC”, extra bases
do not remain in the ssDNA.

"Nt." Nicking endonuclease or restriction
enzymes that leave 3' overhangs are suitable forattaching to the 5' side of insert (attach to reverse
primer of pLSODN PCR template).
For example, at Nt.BspQI site, a single strand iscleaved after “N” indicated by the arrow.
Therefore, extra bases do not remain in the
ssDNA.

Therefore, there are three recommended methods:

1)
2)
3)

5’ end of Insert“Nt” nicking endonuclease“Nt” nicking endonuclease
Restriction enzyme generating 3’ overhang etc.

3’ end of Insert“Nb” nicking endonuclease
Restriction enzyme generating 5’ overhang etc.
“Nb” nicking endonuclease

At least one nicking endonuclease site must be used to give three ssDNA fragments different in size.
If there are no appropriate recognition sites in the DNA fragment of interest because of the existence ofsome nicking endonuclease or restriction enzyme sites in it, some of these sites in the DNA fragment may
need to be changed by site-directed mutagenesis so as not to affect the function of a long ssDNA, forexample silent mutation.
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STEP 2: Design of PCR primer. If the insert length is 3-6 kb, a 13 kb vector backbone is amplified. And if the Insert length is 6-10
kb, a 19 kb vector backbone is amplified by PCR Then the insert is cloned in vector backbone byseamless cloning method. (Figure 1, 2)
Insert3-6kb
6-10kb

Vector backbone
13kb
19kb

■Primer design when insert length is 3-6 kb(13 kb vector backbone is amplified by PCR)
Reverse primer for 13 kb vector backbone:

Forward primer for 13 kb vector backbone:

Reverse primer is complementary strand of:[5’-GTTCGGAGCACTAGGTAAGATGG
TTATGC-3’(3’ end of pLSODN PCR Template)]+ [Nicking Endonuclease or Restriction Enzyme
recognition site]+ [5’ end of insert (20 base)][3' end of insert (20 base)]+ [Nicking Endonuclease or Restriction Enzyme
recognition site]+ [5’-TTCTTACACGTCAGGTGGCACTTT
TCGGG-3’(5’ end of pLSODN PCR Template)]

Example of Forward Primer
Black sequence is Forward primer
5’-TTTTTTTTTTTTTTTTTTTT CATTGC TTCTTACACGTCAGGTGGCACTTTTCGGG-5’
3’-AAAAAAAAAAAAAAAAAAAA GTAACG AAGAATGTGCAGTCCACCGTGAAAAGCCC-3’
enzyme5’ end of
3’ end of insert
pLSODN PCR Template5’----AAACGTGACCCAATG GCTCTTC TTTTTTTT-------------------TTTTTTTT CATTGC TTCTTACACGTCAGGTGGCA----3’
3’----TTTGCACTGGGTTAC CGAGAAG AAAAAAAA-------------------AAAAAAAA GTAACG AAGAATGTGCAGTCCACCGT----5’
enzymeenzymeInsert5’ end of
3’ end ofpLSODN PCR Template
pLSODNPCR Template

Example of Reverse prime
3’ end ofpLSODN PCR Template

5’ end of insert

enzyme5’-GTTCGGAGCACTAGGTAAGATGGTTATGC GCTCTTC TTTTTTTTTTTTTTTTTTTT-3’3’-CAAGCCTCGTGATCCATTCTACCAATACG CGAGAAG AAAAAAAAAAAAAAAAAAAA-5’

Red sequence is Reverse primer

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■Primer design when insert length is 6-10 kb(19 kb vector backbone is amplified by PCR)
Reverse primer for 19 kb vector backbone

Forward primer for 19 kb vector backbone

Reverse primer is complementary strand of:[5’-AATCGAATCCTCACAAACGTGACCCAATG-3’(3’ end of pLSODN PCR Template)]+ [Nicking Endonuclease or Restriction Enzyme
recognition site]+ [5’ end of insert (20 base)]

Forward primer is:[3' end of insert (20 base)]+ [Nicking Endonuclease or Restriction Enzyme
recognition site]+ [5’-TTCTTACACGTCAGGTGGCAC
TTTTCGGG-3’(5’ end of pLSODN PCR Template)]

Example of Forward Primer
Black sequence is Forward primer
5’-TTTTTTTTTTTTTTTTTTTT CATTGC TTCTTACACGTCAGGTGGCACTTTTCGGG-5’
3’-AAAAAAAAAAAAAAAAAAAA GTAACG AAGAATGTGCAGTCCACCGTGAAAAGCCC-3’
3’ end of insert

enzyme5’ end ofpLSODN PCR Template5’----AAACGTGACCCAATG GCTCTTC TTTTTTTT-------------------TTTTTTTT CATTGC TTCTTACACGTCAGGTGGCA----3’
3’----TTTGCACTGGGTTAC CGAGAAG AAAAAAAA-------------------AAAAAAAA GTAACG AAGAATGTGCAGTCCACCGT----5’
enzymeenzymeInsert5’ end of
3’ end ofpLSODN PCR Template
pLSODNPCR Template

Example of Reverse prime
3’ end ofpLSODN PCR Template

5’ end of insert

enzyme5’-AATCGAATCCTCACAAACGTGACCCAATG GCTCTTC TTTTTTTTTTTTTTTTTTTT-3’3’-TTAGCTTAGGAGTGTTTGCACTGGGTTAC CGAGAAG AAAAAAAAAAAAAAAAAAAA-5’

Red sequence is Reverse primer

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PCR AND PURIFICATION OF pLSODN PCR TEMPLATE
The process is PCR, DpnI treatment, electrophoresis, isopropanol precipitation, electrophoresis, andgel extraction.

STEP 3: PCR using pLSODN PCR template
1.2 μl of pLSODN PCR Template is used for 300 μl of PCR reaction. Please use enzyme suitable forlong amplicons such as PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.). Inappropriate enzyme
may not work well.

STEP 4: Treatment by DpnI
1.
2.
3.

Add 60 units of DpnI to 300 μl of PCR product.
Incubate at 37℃for 1 hour.
Incubate at 80℃for 20 minutes.

STEP 5: Electrophoresis
Electrophorese at 100V for 40 minutes using 0.8 % agarose gel with 1×TAE to check the size of PCR
product.*1, *2, *3
One μl of PCR product in STEP 3 is normally enough to recognize the band.
If too much DNA is loaded, DNA may remain in the wells or the bands may smear. Reducing theconcentration of DNA may improve this problem.
*3 The composition of 1×TAE buffer is 40 mM Tris, 20 mM acetic acid and 1 mM EDTA (ref. 2).
*1*2

STEP 6: Isopropanol precipitation1.

Add 1/10 volume of 3M sodium acetate (pH 5.2), 1/50 volume of 5 mg/ml glycogen, 0.7 volumeof isopropanol.
2. Mix and centrifuge at 20,000×g for 30 min at 4℃
3. Discard the supernatant.
4. Wash DNA pellet with 1 ml of 70% ethanol with vortexing.
5. Centrifuge at 20,000×g for 15 min.
6. Discard the supernatant.
7. Wash DNA pellet with 1 ml of 70% ethanol with vortexing
8. Centrifuge at 20,000×g for 15 min.
9. Discard the supernatant
10. Air-dry the pellet
11. Add 10μl of TE *1*1

For large size DNA, it may take some time to dissolve the precipitate. Please incubate for about 30 minutesat room temperature until precipitation dissolve completely. There may be a thin precipitate spreading on thewall of the tube.

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STEP 7: Electrophoresis and gel extraction
1.
2.
3.
4.

Prepare 0.8% agarose gel by adding 40 μl of Crystal Violet Solution to 100 ml of melted agarose
in 1×TAE. *1
Add 30 μl of Denaturing Gel-Loading Buffer to 10 μl of PCR product dissolved in TE (STEP 6). *2
Subject the mixture to 0.8% agarose gel electrophoresis in 1×TAE including Crystal Violet (add 40
μl Crystal Violet Solution / 100 ml TAE). Run at 100 V constant voltage for 40 minutes. *3, *4
Purify the DNA fragment from the excised gel using commercially available kit (e.g. Monarch
DNA Gel Extraction Kit, New England Biolabs Inc.).

The DNA band in Crystal Violet-containing gel can be directly seen under ambient light.
If standard Loading Dye is added, large amounts of high molecular weight DNA may be left in the wells. In
this step Denaturing Gel-Loading Buffer is necessary. Unlike separation of ssDNA (STEP 16, 17), the sample
is not heated before electrophoresis.
*3 In the electrophoresis with Crystal Violet, the migration of DNA is influenced by the DNA amount in eachband. Therefore molecular weight marker does not necessarily migrate to the expected position. However,DNA band in the gel including Crystal Violet can be directly seen in real time. So the band to excise can be
identified by comparison with the result of analytical electrophoresis (STEP 5).
*4 Crystal Violet concentration in the running buffer and gel gradually decrease. Check the color of the gel andrunning buffer, and if needed, add Crystal violet into the running buffer on the anode side during
electrophoresis.
*1
*2

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CLONING OF INSERT INTO pLSODN PLASMID
STEP 8: Preparation of insert
Prepare the fragment of insert as appropriate.

STEP 9: Ligation and transformation
1.

Ligate insert (STEP 8) and purified pLSODN PCR product (STEP 7) with commercially available
seamless cloning kit (NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs Inc.) is
recommended).
Transformation it to appropriate Competent Cell. *1

2.
*1

For large plasmids, transformation efficiency and ligation efficiency may be decreased, so competent cellswith high competency is recommended.

STEP 10: Colony PCR
The following sequence can be used as colony PCR primer. However, in some case, PCR may not work
well depending on the sequence of insert.
For 19 kb vector backbone
19 kb Forward Primer: TTTGTATGGCAAGACATCATGGGCGTTAAC
19 kb Reverse Primer: TGTTGAATACTCATACTCTTCCTTTTTCAAThis primer combination gives “insert + about 350 bp”.

For 13 kb vector backbone
13 kb Forward Primer: AACAGGTAAGACTACCCGTTGGGAGAAAGA
13 kb Forward Primer: TTCGATGTAACCCACTCGGGCACCCAACTG
This primer combination gives “insert + about 500 bp”.

STEP 11: Plasmid purification
Culture the selected clone using LB medium (100 μg/ml Ampicillin), SOB medium (100 μg/ml
Ampicillin) etc. *1
Purify the plasmid with a purification kit (e.g. QIAGEN Plasmid Kit (QIAGEN N.V.)).

1.
2.
*1

The plasmid of STEP 11 is large (16-29 kb). Mutation tends to be easily introduced into large plasmids, so
multiple subculture should be avoided as much as possible.

STEP 12: Confirmation of insert size by restriction enzyme
There are restriction enzymes (double strand break) whose recognition site is the same as nicking
endonucleases. Their name are the “name of the nicking endonuclease” minus “Nt.” or "Nb." (e.g. Nicking
Endonuclease: “Nt.BspQI”, Restriction Enzyme: “BspQI”). Using these restriction enzymes, the insert
length can be checked.

STEP 13: Sequencing of insert
The sequence of insert should be confirmed before preparing the ssDNA.

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DIGESTION OF THE PLASMID WITH NICKING ENDONUCLEASE AND
EXTRACTION OF LONG ssDNA
The plasmid harboring your DNA fragment of insert (STEP 13) is digested with two nicking
endonucleases or combination with nicking endonuclease and restriction enzyme. After digestion, thenicked plasmid solution must be desalted with ethanol precipitation. Desalting of nicked plasmid
solution is very important for agarose gel electrophoresis of ssDNA because remaining salt and Mg2+
stabilize the dsDNA and prevent complete denaturation of the nicked plasmids. One example of theseprocedures is shown below.

STEP 14: Digestion of plasmid with nicking endonuclease
The plasmid harboring your DNA fragment of interest (STEP 13) is digested with two nicking
endonucleases or combination with a nicking endonuclease and restriction enzyme. *1, *2*1*2

From 100 μg of plasmid, about 2-6 μg of ssDNA is often obtained.
Reaction condition (Buffer, amount of enzyme and reaction time etc.) are in accordance with manufacturer’s
instructions.

(Example)
plasmid (100 μg)
10x 3.1 NEBuffer
Nb.BbvCI (10 unit/μl)
Nt.BspQI (10 unit/μl)
dH2O
Total volume
Incubate at 37℃ for 3 hr.

variable
10 μl
5 μl
5 μl
variable
100 μlSTEP 15: Desalting
1.

2.
3.
4.

Add 1/10 volume of 3M sodium acetate (pH 5.2), 1/50 volume of 5 mg/ml glycogen, 2.5 volumes of
ethanol.
Mix and centrifuge at 20,000×g for 30 min at 4℃.

Discard the supernatant.
Centrifuge for another minute, collect the remaining liquid and remove with a micropipette.
*1
Wash the DNA pellet with 1 ml of 70% ethanol with vortexing. *2
5. Centrifuge at 20,000×g for 15 min.
6. Discard the supernatant.
7. Centrifuge for another minute, collect the remaining liquid and remove with a micropipette.Wash
the DNA pellet with 1 ml of 70% ethanol with vortexing*3, *4
8. Centrifuge at 20,000×g for 15 min.
9. Discard the supernatant.
10. Centrifuge for another minute, collect the remaining liquid and remove with a micropipette.
11. Air-dry the pellet
12. Add 100μl of TE (The resulting final concentration of nicked plasmid was 1 μg/μl). *5*1*2

To exclude salt, the residual liquid must be removed as much as possible.
The precipitate is detached by vortexing and floats violently in 70% ethanol, but there is no need to worry
about re-dissolution. As desalting proceeds, the precipitate becomes viscous.
*3 Wash twice with 70% ethanol in order to desalt sufficiently.
*4 If the nicked plasmid solution contains excessive salt or Mg+2, it cannot be completely denatured even by
mixing with the Denaturing Gel-Loading Buffer. Desalting of the nicked plasmid solution is required.
*5 For large size DNA, it may take some time to dissolve the precipitate. Please incubate for about 30 minutesat room temperature until precipitation dissolve completely. There may be a thin precipitate spreading on thewall of the tube.

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STEP 16: Analytical agarose gel electrophoresis
The generation of ssDNA from the nicked plasmid (STEP 15) is analyzed with agarose gelelectrophoresis.
The agarose concentration is determined as follows according to the insert length.
Length of Insert
Concentration of Agarose
3-6 kb
1.0%
6-10 kb
0.8%
1.
2.
3.
4.
5.
6.

Prepare loading samples by mixing the nicked plasmid (STEP 15) (300 ng) and 3 volumes of the
Denaturing Gel-Loading Buffer.*1, *2
Heat the mixture at 70°C for 5 min. *3
Chill on ice for 1 min.
Subject the mixture to agarose gel electrophoresis with 1×TAE. Run at 100 V. *4, *5, *6
Stain the gel with 0.5 μg/ml of ethidium bromide for 20 min.
Examine the gel on a UV illuminator (figure 3).

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. Desalting of the nicked plasmid solution is required.
*2 After mixing with the Denaturing Gel-Loading Buffer, the concentration of the nicked plasmid in theloading sample should be under 0.25 μg/μl.
*3 After heating and chilling, quickly load the samples into wells of the gel and promptly start electrophoresis.
The denatured DNA is gradually reannealed even in Denaturing Gel-Loading Buffer. Furthermore, once thesamples are loaded into the wells, dilution of the Denaturing Gel-Loading Buffer in the sample solution could
occur gradually.
*4 The approximate size of ssDNA can be estimated by “λ / HindIII DNA for long ssDNA preparation kit”
(electrophoresis after incubating at 70 °C for 5 minutes and on ice for 1 minute). The mobility of ssDNA is morechangeable depending on the sequence than dsDNA, therefore this λ / HindIII DNA indicates rough size.
*5 23130 bases and 6557 bases of the “λ / HindIII DNA for long ssDNA preparation kit” may appear as doubleband.
*6 Commercially available DNA molecular weight markers sometimes could not give clear bands under mixing
with Denaturing Gel-Loading Buffer. It may be caused by different migration of complementary strands due totheir secondary structure or incomplete denaturation.
1
2
3
4
5
6
7
*1

bases
23130
9416
6557
4361
2322
2027

Figure 3. Analytical agarose gel electrophoresis of the nicked plasmid
Lane 1: λ/HindIII DNA for long ssDNA preparation kit (aliquoted 20 μl into new tube, heat and chill before loading)Land 2: Uncut plasmid
Lane 3: Purified pLSODN PCR product (STEP 7)
Lane 4: Insert prepared for cloning (STEP 8)
Lane 5: Nicked plasmid (digested by nicking endonuclease and restriction enzyme)
Lane 6: Uncut plasmid
Lane 7: Nicked plasmid (digested by two nicking endonucleases)
Samples of Lane 2-7 were added to 3 volumes of Denaturing Gel-Loading Buffer and incubated at 70 ° C for 5 minutes
and on ice for 1 minute before electrophoresis.
The blue, orange and red arrows show your interest ssDNAs, linear single-stranded vector DNAs and circular singlestranded whole plasmid DNAs, respectively. The yellow arrow shows randomly broken circular single-stranded wholeplasmid DNAs (They originate in open circular form of plasmid DNA).

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STEP 17: Preparative agarose gel electrophoresis
The nicked plasmid is subject to agarose gel electrophoresis for ssDNA extraction.
The agarose concentration is determined as follows according to the insert length.
Length of Insert
Concentration of Agarose
3-6 kb
1.0%
6-10 kb
0.8%
1.
2.
3.
4.
5.
6.

7.
8.
9.

Cool the 1×TAE to be used for electrophoresis on ice or in a refrigerator.
Prepare the agarose gel by adding 40 μl of Crystal Violet Solution to 100 ml of melted agarose in
1×TAE. *1
Prepare loading samples by mixing the nicked plasmids (STEP 15) and 3 volumes of the
Denaturing Gel-Loading Buffer.*2, *3
Heat the mixture at 70°C for 5 min.*4
Chill on ice for 1 min.
Put the electrophoresis apparatus on ice in an ice bucket in a draft chamber. Subject the mixture toagarose gel electrophoresis in pre-chilled 1×TAE with crystal violet (add 40 μl Crystal Violet Solution/ 100 ml TAE). *5, *6, *7, *8
Run at 100 V, low temperature (less than 20°C)
Monitor the ssDNA blue bands moving in the gel during electrophoresis. Stop the electrophoresis
after the bands are sufficiently resolved (Figure 4). *9, *10, *11
Excise the ssDNA band from the gel. *12

ssDNA band in crystal violet containing gel can be directly seen under ambient light.
Desalting of the nicked plasmid solution is important.
To get a highly purified long ssDNA, the long ssDNA of interest should be loaded greater than 0.5 μg/band.
At the same time, the concentration of the nicked plasmid in the loading sample should be under 0.25μg/μl after mixing with the Denaturing Gel-Loading Buffer.
*4 The heating and cooling procedure of the nicked plasmid should be carried out just before loading into wellsof the gel. After loading, quickly start electrophoresis.
*5 Do not load a sample too much in a well because it may overflow and cause contamination. It is
recommended to make deep wells with thick gel.
*6 For the safe use of Crystal Violet, electrophoresis with Crystal Violet should be carried out in a draft
chamber. Do not do electrophoresis with Crystal Violet or ethidium bromide in a closed space (especially in acold room). These dyes diffuse into the air with the water vapor or bubbles generated by electrolysis during
electrophoresis.
*7 Electrophoresis at a low temperature is recommended (less than 20 °C). It gives a better resolution andstrong Crystal Violet staining. If the temperature of gel and running buffer exceeds 25 °C, Crystal Violet
staining of lssDNA 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.
*8 To keep the gel temperature low during electrophoresis in a draft chamber, we recommend placing theelectrophoresis apparatus on ice in an ice bucket. At least, use a pre-chilled 1xTAE running buffer on ice tokeep a low temperature.
*9 100 ng of ssDNA band is sufficient for detection with Crystal violet staining.
*10 In the electrophoresis with Crystal violet, the migration of ssDNA is influenced by DNA amount in eachband. Therefore molecular weight marker is not necessarily migrated to the expected position. However,
ssDNA band in the gel including crystal violet can be directly seen in real time. So the band to excise can be
identified by compared with the result of analytical electrophoresis (STEP 16) (Figure 3, 4).
*11 Crystal violet concentration in running buffer and gel gradually decrease. Check the color of gel andrunning buffer, and if needed, add Crystal violet into the running buffer on the anode side during
electrophoresis.
*12 Minimize the size of the gel slice by removing extra agarose.
*1
*2
*3

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1

2

3

10 kb ssDNA

Figure 4. Preparative agarose gel electrophoresis.
Lane 1, 2, 3: digested plasmid
The photo was taken under white light.

STEP 18: Extraction of long ssDNA
Before start:• Add 45 ml of 100 % ethanol to 8 ml of Wash Buffer 1 and 11 ml of Wash Buffer 2.
• Isopropanol is required.
• All centrifugation steps should be carried out at 16,000×g (around 13,000 rpm in a conventional
microcentrifuge) at room temperature (20℃- 25℃). Centrifugation at lower temperature might
affect the long ssDNA yield.
1.
2.
3.

Transfer the gel slice to a 1.5 ml microcentrifuge tube and weight the gel slice (STEP 17).
Add 3 volumes of Gel Dissolving Buffer.
Incubate the tube at 50°C, vortexing periodically until the gel slice is completely dissolved for 1015 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 theCollection 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 Buffer1 to the Spin Column and centrifuge for 1 min. Discard the flowthrough in the Collection Tube with a 1ml pipette tip (1st Wash).
9. Repeat wash with Wash Buffer 1 (Step10, 2nd Wash).
10. Add 500 μl of Wash Buffer 1 to the Spin Column, close the cap tightly and vortex for 5 seconds towash the whole 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 towash both the 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.
13. Add 500 μl of Wash Buffer 2 to the Spin Column and centrifuge for 1 min. Discard the flowthrough in the Collection Tube with a 1ml pipette tip (4th Wash).
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. Immediately, centrifuge the Spin Column for 1 min to elute long ssDNA. *7*1

Dissolve the agarose completely. If not, it might decrease the recovery yield of long ssDNA and cause
contamination of agar or buffers.
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If the volume of dissolved sample exceeds 500 μl, the loading of the sample onto the column should be
performed in multiple rounds so as not to exceed 500 μl.
*3 Do not flip the Collection Tube to discard the flow-through. It contaminates the edge and upper innerwall of the Collection Tube. Use a 1 ml pipette tip to discard the flow-through.
*4 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 included in the kit. They can be purchased from many companies (e.g. Corning Axygen #MCT-200-NC).
*5 Do not wash the Spin Column with Gel-Dissolving Buffer only or its dilution.
*6 Typical elution volumes are 15 μl -40 μl. Water can be used to elute the long ssDNA. The average elution
volume is 36.5 μl from 40 μl Elution Buffer volume, and 12 μl from 15 μl.
*7 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 spincolumn (e.g. Merck Ultrafree-MC GV 0.22 μm).
*2

1

2

3

10 kb ssDNA

Figure 5. Agarose gel electrophoresis of purified long ssDNA
Lane 1: Uncut plasmid
Lane 2: Digested plasmid
Lane 3: Purified long ssDNA (10 kb fragment)

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Troubleshooting Guide, FAQ
TROUBLEThe ssDNA band
does not clearly
separate during
electrophoresis.

POSSIBLE CAUSESOLUTION
Desalting of the nicked plasmid solution is very
important.
ssDNA bands may not separate clearly if salt isremaining.
Wash thoroughly with 70% ethanol in ethanol precipitation.
Specifically, washing with 70% ethanol twice, vortex at
washing. After removing the supernatant by sedimentation or
washing operation, centrifuge again to collect the residual liquidon the inner wall surface and remove it thoroughly with amicropipette.

Salt remaining

Desalting of the nicked plasmid solution is very
important.
ssDNA bands may not separate clearly if salt isremaining.
Wash thoroughly with 70% ethanol in ethanol precipitation.
Specifically, washing with 70% ethanol twice, vortex at
washing. After removing the supernatant by sedimentation or
washing operation, centrifuge again to collect the residual liquidon the inner wall surface and remove it thoroughly with amicropipette.

Salt remaining
Low ssDNA yield

Low yield ofnicked plasmidin ethanol precipitation

Is it possible
to prepare ssDNA
smaller than 3 kb
using this kit?

As in the above protocol, adding glycogen during
ethanol precipitation may increase DNA recovery.

In principle, it is also possible to prepare ssDNA smaller than 3 kb with this kit, Long ssDNA
Preparation Kit for 10kb (Cat. No. DS635).
However, "Long ssDNA Preparation Kit for 1.5kb (Cat. No. DS615)" and "Long ssDNA
preparation kit for 3.0kb (Cat. No. DS625)" are more suitable for the preparation of ssDNA
smaller than 3 kb. We recommend you to use #DS615 or #DS625.
In order to obtain the same amount of ssDNA with #DS635 as compared with #DS615 or#DS625, a larger amount of plasmid is needed. Also, as the concentration of plasmid to apply
to electrophoresis gel is limited (about 0.25 μg/μl), the ssDNA concentration in the band also
decreases, which may affect the purification degree and yield.

References
1. 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.
2. 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
DS612
DS650
DS670
DS615
DS625

Denaturing Gel-Loading Buffer
1 ml x 5
Denaturing Gel-Loading Buffer
1 ml x 2
Long ssDNA Gel Extraction Kit for 10kb
λ/HindIII DNA for long ssDNA preparation kit
Long ssDNA Preparation Kit for 1.5 kb
Long ssDNA Preparation Kit for 3.0kb

Purchaser Notification
The product shall be used by the purchaser for internal research purpose only. Biodynamics laboratory
Inc. has pending patent applications related to the product. For information concerning licenses for18

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commercial use, contact Biodynamics laboratory Inc. The products and plasmids that are modified or
harboring a DNA may not be transferred to third parties, resold, modified for resale, or used to
manufacture commercial products or to provide a service to third parties without prior written approvalof Biodynamics laboratory Inc.

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