Long ssDNA Preparation Kit for 1.5kb

Product#: DS615
$700.00

Type:

  • Academic
  • Commercial
Availability:
Ships in 1-2 weeks

Long ssDNA Preparation Kit for 1.5kb (Academic / Commercial Entities)

A simple and highly effective method for preparing a long single stranded DNA for CRISPR workflows.  
Knock-in efficiency becomes dramatically better when CRISPR/Cas9 is performed with long single-stranded DNA.
The long ssDNA prepared by our kits has a defined sequence and length with high fidelity enzymes and does not include mutation and terminal deletion caused by PCR.


Cat. No. DS615
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
  • Yoshimi K. et al., ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Commun. 2016 Jan 20;7:10431 
  • Wang Y.et al., Long noncoding RNA lncHand2 promotes liver repopulation via c-Met signaling. J Hepatol. 2018 Oct;69(4):861-872.
  • Zhu P. et al., LncGata6 maintains stemness of intestinal stem cells and promotes intestinal tumorigenesis. Nat Cell Biol. 2018 Oct;20(10):1134-1144.
  • Zhu P. et al., IL-13 secreted by ILC2s promotes the self-renewal of intestinal stem cells through circular RNA circPan3. Nat Immunol. 2019 Feb;20(2):183-194.
  • Nozaki S, and Niki H. Exonuclease III (XthA) enforces in vivo DNA cloning of Escherichia coli to create cohesive ends. J Bacteriol. 2018 Dec 10.
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