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Long single stranded DNA preparation

Long ssDNA Preparation Kit (LsODN Preparation Kit)

Highlights

  • A long ssDNA (within 10,000 bases) can be prepared.

  • A long ssDNA has defined sequence and length.

  • Simple principle and easy procedure.

  • High yield and high quality.

The Long ssDNA (LssDNA/LssODN) Preparation Kits give a simple and easy method for generation of a long single stranded DNA (within 10,000 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, 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.

Principle

  1. The DNA of interest is cloned into a plasmid between the two Nicking enzyme sites, or between the Nicking enzyme site and the restriction enzyme site.

  2. 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.

  3. The nicked plasmid is denatured and then subjected to agarose gel electrophoresis.

  4. The band corresponding to a long ssDNA is excised and extracted.

Long ssDNA Gel Extraction Kit

Long ssDNA Gel Extraction Kit has been optimized for extraction of high-quality Long ssDNA from agarose gel.
There are two lineups according to ssDNA length.

Long ssDNAs prepared by this kit

Comparison with other companies’ products


Comparison of our gel extraction kit (DS640 & DS650) with kits A, B, and C of other companies.

After separation of lssDNA (2 μg) of various lengths by agarose gel electrophoresis, gel pieces containing the target bands were cut out and extracted and recovered (40 μl elution) using each company’s kit. From the 40 μl of each lssDNA solution recovered, 16 μl was taken, diluted to 100 μl, and the absorption spectrum was measured (upper figure). Similarly, 5 μl was taken from each lssDNA solution and subjected to agarose gel electrophoresis (bottom figure). The results of both experiments showed that the highest recovery (79%-93%) of lssDNA of any length was obtained using our kit. The results in the figure above indicate that the lssDNA prepared with kits A and B contains GuSCN, a gel dissolving agent, making accurate quantification by absorption spectrophotometry difficult. On the other hand, the lssDNA prepared by kit C does not appear to be contaminated with the gel dissolving agent, but it is necessary to note that the contamination cannot be detected in the absorption spectrum because NaI is used as the gel-dissolving agent. The agarose gel electrophoresis results (see figure below) not only show the high recovery rate of our kit, but also indicate that the degree of degradation is minor, especially for the longer 6Kb and 10kb lssDNAs.

Manuscript using this product

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.

Code

Size

1kit

Manuals

SDS

Code

Size

1kit

Manuals

SDS

Code

Size

1kit

Manuals

SDS

To access the full sequences of these kits, you need password. The password is shown in the inner lid of the box.

Code

DS611

Product Name

Denaturing Gel-Loading Buffer

Size

1mlx5

Manuals

SDS

Code

DS612

Product Name

Denaturing Gel-Loading Buffer

Size

1mlx2

Manuals

SDS

Code

Size

600μl
(30Loadings)

Manuals

SDS