Long ssDNA Preparation 1.5-3kb (Academic/Commercial)

Description:

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.

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.

Plasmid Map

Data 


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.