No More Sales-Long ssDNA Preparation Kit for 3.0kb
Long ssDNA Preparation Kit for 3.0kb (LsODN Preparation Kit)
Cat. No. DS620
Size 1 Kit
Storage Conditions
Plasmids and Denaturing Gel-Loading Buffer should be stored at -20℃. DynaMarker® Prestain Marker for RNA High should be stored at -80℃ directly from a dry ice shipping box.
Description
Single-stranded DNA (ssDNA) is widely used for molecular biology and biotechnology 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 (LsODN Preparation Kits) give a simple and easy method for generation of a long ssDNA (pLSODN-1 and -2D for ssDNA fragments within 1,500 base and pLSODN-3 and -4D for ssDNA fragments within 3,000 base).
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, not high-fidelity reverse transcriptase reaction or not high-fidelity synthetic oligonucleotides. The procedure of this method is almost the same as the method to obtain dsDNA fragments. The DNA of interest 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 gel electrophoresis. The band corresponding to a long ssDNA is excised and extracted with commercially available kits.
Long ssDNA Preparation Kit (LsODN Preparation Kit) PAT.P
The Long ssDNA Preparation Kits (LsODN Preparation Kits) give a simple and easy method for generation of a long single stranded DNA (within 1,500 base or within 3,000 base). 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, not high-fidelity reverse transcriptase reaction or not high-fidelity synthetic oligonucleotides.
The procedure of this method is almost the same as the method to obtain dsDNA fragments. The DNA of interest 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 gel electrophoresis. The band corresponding to a long ssDNA is excised and extracted with commercially available kits.
- A long ssDNA (within 1,500 base or within 3,000 base) can be prepared.
- A long ssDNA has defined sequence and length.
- Simple principle and easy procedure.
- High yield and high quality.
- The DNA of interest is cloned into a plasmid using a pair of two nicking endonuclease sites or a combination of a nicking endonuclease site and a restriction enzyme site
- The resulting plasmid harboring the DNA is digested with a pair of nicking endonucleases or a combination of a nicking endonuclease and a restriction enzyme
- The nicked plasmid is denatured using by Denaturing Gel-Loading buffer (DS611) and then subjected to agarose gel electrophoresis
The resulting plasmids were digested with Nt.Bsp QI and the Nb. Bsr DI.
The double nicked plasmid was mixed with Denaturing Gel-Loading buffer (DS611) and heated, then loaded to conventional non-denaturing agarose gel electrophoresis.
The band corresponding to a long ssDNA is excised and extracted.
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Fig. Long ssDNAs prepared by this kit A 1.5 kb DNA fragment of interest was cloned between the Nt.BspQI and the Nb.BsrDI sites of pLSODN-1. Similarly, a 3.0 kb DNA fragment of interest was cloned between the Nt.BspQI and the Nb.BsrDI sites of pLSODN-3. The resulting plasmids were digested with Nt.BspQI and Nb.BsrD. The double nicked plasmid was mixed with Denaturing Gel-loading Buffer and heated, then loaded to conventional non-denaturing agarose gel electrophoresis. The band corresponding to a long ssDNA was excised and extracted. Lane :1 The double nicked pLSODN-1 harboring 1,500 bp DNA fragment Lane :2 Purified long ssDNA(1,500 base) Lane :3 The double nicked pLSODN-3 harboring 3,000 bp DNA fragment Lane :2 Purified long ssDNA(3,000 base) |
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, SYBER® Green, crystal violet or any other DNA staining reagent
• DNA gel extraction kit (e.g. QIAquick® Gel Extraction Kit)
For Effective Knock-In by Genome EditingLong Single Strand DNA Preparation Kit
The Long ssDNA Preparation Kits (LsODN Preparation Kits) provide a simple and easy method for generation of a long ssDNA (within 1,500 base or within 3,000 base). A long ssDNA prepared by this kit has defined sequence and length as it does not include inside mutation and terminal deletion caused by PCR, exonuclease side reaction, not high-fidelity reverse transcriptase reaction or not high-fidelity synthetic oligonucleotides.
The procedure is almost same as the method to obtain dsDNA fragments. The DNA of interest 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 gel electrophoresis. The band corresponding to a long ssDNA is excised and extracted with commercially available kits.
Kit Contents
DS610 Long ssDNA Preparation Kit for 1.5 kb (LsODN Preparation Kit)
- pLSODN-1 10 µg (0.5 µg/µl)
- pLSODN-2D 10 µg (0.5 µg/µl)
- Denaturing Gel-Loading Buffer 1 ml (100 loadings)
- DynaMarker® Prestain Marker for RNA High 90 µl (18 loadings)
DS620 Long ssDNA Preparation Kit for 3.0 kb (LsODN Preparation Kit)
- pLSODN-3 10 µg (0.5 µg/µl)
- pLSODN-4D 10 µg (0.5 µg/µl)
- Denaturing Gel-Loading Buffer 1 ml (100 loadings)
- DynaMarker® Prestain Marker for RNA High 90 µl (18 loadings)
Features
- A long ssDNA (within 1,500 base or within 3,000 base) can be prepared.
- A long ssDNA has defined sequence and length.
- Simple principle and easy procedure.
- High yield and high quality.
Related Item
DynaMarker, Prestain Marker for RNA High (cat. no. DM260)
Reference
- ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T. (2016) Nature Communications. 20;7:10431
