Product Name : Long ssDNA Gel Extraction Kit for 10kb
Code No. : DS650

日本語データシート

This product is for research use only

Kit Components: 25 reactions
Components
Crystal Violet Solution
Gel-Dissolving Buffer
Wash Buffer 1
Wash Buffer 2
Elution Buffer
Spin Column

Contents
Crystal Violet 4mg/ml (2,500 x).
It contains guanidine thiocyanate as chaotropic salts.
Tris-base buffer(Add 45 ml of 100 % ethanol to the Wash Buffer before use.)
Tris-base buffer(Add 45 ml of 100 % ethanol to the Wash Buffer before use.)
10 mM Tris-HCl, pH 8.0.

Size5 ml45 ml
8 ml
11 ml
5 ml
25 pieces• Ethanol and isopropanol are required for the product.
• If precipitated material has formed in Gel-Dissolving Buffer, heat to dissolve at 37℃.
• Heat block are required for 50-70℃ incubation.
• Denaturing Gel-Loading Buffer is required for the agarose gel electrophoresis of long ssDNA. It is not included inthe kit. It can be purchased individually from BioDynamics Laboratory Inc. (see “Related Products“ on page 4).

Introduction: The Long ssDNA Gel Extraction Kit is a highly-specialized kit for long ssDNApurification from agarose gel. The spin column, buffer formulations and purification protocol are
optimized for high recovery yields and high purity of long ssDNA. Long ssDNA purified with the kit ishigh-quality, with low mechanical degradation and without ultraviolet light-induced damage from thepurification 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 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 - 10,000 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.

Storage conditions: Stable at 15 to 25℃ for 24 months from the date of receipt.
Caution!

Wear gloves and protective clothing while handling the Crystal Violet.

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Protocol of DNA excision and purification:
STEP 1: Preparation of agarose gel containing Crystal Violet
An example of this is given for making 100 ml of a 0.8% agarose gel
1.

Add 0.8 g of agarose powder to 100 ml of 1×TAE buffer and mix them.

2.

Dissolve the 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.

STEP 2: 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) onice or in a refrigerator. *1

2.

Put the horizontal gel electrophoresis apparatus on ice in an ice bucket in a draft chamber. *2, *3, *4

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 theDenaturing Gel-Loading Buffer. *6, *7, *8
6. Heat the mixture at 70°C for 5 min, then chill on ice for 1 min. Subject the mixture to agarose gelelectrophoresis. *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
9. Excise the long ssDNA band of interest from the agarose gel with a scalpel.*13, *1412Fig 1. Agarose gel electrophoresis using the gel containing Crystal Violet.
0.8% agarose gel in 1x TAE, 5.5cm x 6.0 cm. The loading volume of each sample is 24 μl.
Lane 1: pLSODN ( 6 kb Fragment), double-nicked and denatured *15
6 μg
Lane 2: pLSODN (10 kb Fragment), double-nicked and denatured *15 6 μg

10 kb (1μg)
6 kb (1μg)*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.

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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.
*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 μlof 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 Denaturing Gel-Loading Buffer is not included in the kit. It can be purchased individually from BioDynamics
Laboratory Inc. (see “Related Product “on page 4)*7 Desalting of the long ssDNAs sample (or the nicked plasmid) solution in advance is important for agarose
gel electrophoresis. If the long ssDNAs sample (e.g. the nicked plasmid) solution contains excessive salt andMg+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 ofresidual salt in the long ssDNAs sample (or the nicked plasmid) solution. We recommend removing the salt inthe long ssDNAs sample through ethanol precipitation. Importantly, wash with 70% ethanol twice, vortex
vigorously during washing. In ethanol precipitation and washing operations, the supernatant is usually simply
removed after centrifugation, but to ensure thorough desalting, it should be further centrifuged again afterward tocollect residual liquid adhering to the wall surface and remove it thoroughly with a micropipette.
*8 To get a highly purified long ssDNA, the long ssDNA of interest should be loaded greater than 0.5μg/band. At
the same time, the concentration of the nicked plasmid in the loading sample should be under 0.25 μg/μl
after mixing with the Denaturing Gel-Loading Buffer.
*9 The heating and cooling procedure of the long ssDNAs sample (e.g. the nicked plasmid) mixture should be
carried out just before loading into wells of the gel.
*10 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.
*11 Long ssDNA bands moving in Crystal Violet-containing gel can be directly seen under ambient light inreal time during electrophoresis. The electrophoresis can be stopped as soon as the bands are sufficientlyresolved.
*12 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 isadded to 250ml running buffer on the anode side every 30 min at 100V constant voltage).
*13 The long ssDNA band can be visible as a blue band and can be easily excised from the agarose gel on thebench.
*14 Minimize the size of the gel slice by removing extra agarose.
*15 The pLSODN (6 kb Fragment) and pLSODN (10 kb Fragment) were constructed by inserting a 6 kb fragment
and a 10 kb fragment of DNA into cloning site of 13 kb plasmid and 20 kb plasmid, respectively. The doublenicked plasmids were obtained by double digestion with Nt.BspQI and Nb.BbvCI. The double-nicked ThepLSODN (6 kb Fragment) gives mainly two linear ssDNAs (6 kb and 13 kb) and one circular ssDNA (19 kb).
Similarly, the double-nicked The pLSODN (10 kb Fragment) gives two linear ssDNAs (10 kb and 20 kb) and
one circular ssDNA (30 kb).

DNA f

STEP 3: 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 13,000 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

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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). *3, *4
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, *8123Figure 2. Agarose gel electrophoresis of purified long ssDNAs.

40.8% agarose gel in 1x TAE. 5.5cm x 6.0 cm.
Lane 1: pLSODN ( 6 kb Fragment), double-nicked and denatured
Lane 2: a long ssDNA purified from pLSODN (6 kb Fragment)
Lane 3: pLSODN (10 kb Fragment), double-nicked and denatured
Lane 4: a long ssDNA purified from pLSODN (10 kb Fragment)

800 ng
400 ng
800 ng
400 ng

10 kb (0.4μg)
6 kb (0.4μg)

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*1*2*3*4*5*6*7*8Dissolve 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 anew Collection Tube at several washing steps to avoid contamination. However, spare collection tubes are notincluded in 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 15μl -40μl. 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.
The total yield of ssDNA recovery from a 12 μg of a nicked plasmid pLSODN (6 kb Fragment) or pLSODN(10kb Fragment) was about 1.2 μg (100% recovery is 2 μg) using one column of the Long ssDNA GelExtraction Kit for 10 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).

Related Products:
DS611
DS612
DS615
DS625
DS635DS650

Denaturing Gel-Loading Buffer
Denaturing Gel-Loading BufferLong ssDNA Preparation Kit for 1.5 kb
Long ssDNA Preparation Kit for 3.0 kb
Long ssDNA Preparation Kit for 10 kb
Long ssDNA Gel Extraction Kit for 10 kb

1 ml x 5
1 ml x 2

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Ver.1.2