Long ssDNA Preparation Kit for 10kb, Commercial Entities DS635C
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Long ssDNA Preparation Kit for 10kb, Commercial Entities DS635C

Price: $1,760.00
  • Item #: DS635 C
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Long ssDNA Preparation Kit for 10kb, Commercial Entities

Cat. No.  DS635 C
Store RT,-20°C
Size 1 kit

 

Description

The Long ssDNA(Long Single Strand DNA) 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 our kit (#DS640) specially designed for this kit.


Kit Contents

  • DS615 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)
  • Long ssDNA Gel Extraction Kit for 3kb (DS640) (25preps)
  • DS625 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)
  • Long ssDNA Gel Extraction Kit for 3kb (DS640) (25preps)

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.

Principle

  1. 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.
  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.
DS615_Fig1.jpg



Plasmid Map

DS615_Fig2.jpg



Data 

DS615_Fig3.jpg
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 1.5 kbp DNA fragment
Lane 2: Purified long ssDNA (1.5 kb)
Lane 3: The double nicked pLSODN-3 harboring 3.0 kbp DNA fragment
Lane 4: Purified long ssDNA (3.0 kb)


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) 



 

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