[LsODN Preparation Kits] Long Single Strand DNA Preparation Kit [DS610, DS620]_BioDynamics laboratory_Funakoshi - 코아사이언스

Long Single Strand DNA Preparation Kit

For Effective Knock-In by Genome Editing

Cited in Nature Communications, 2016!

간단한 과정을 통해 높은 순도로 목적 서열을 갖는 긴 사슬의 ssDNA (<1,500 base 또는 < 3,000 base)를 얻을 수 있는 키트입니다. 기존 준비 방법의 단점으로 지적되는 PCR, exonuclease side reaction, low-fidelity reverse transcriptase reaction 또는 low-fidelity synthetic oligonucleotides에 의한 내부 돌연변이와 말단 부위 결손에 대한 염려가 없습니다.

The Long ssDNA Preparation Kits (LsODN Preparation Kits) provide a simple and easy

method for generating of a long ssDNA (<1,500 base or < 3,000 base).

A long ssDNA prepared by this kit has a defined sequence and length as it does not include internal

mutation and terminal deletion caused by PCR, exonuclease side reaction, low-fidelity reverse

transcriptase reaction or low-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

by a pair of two nicking endonucleases or a combination of a nicking endonuclease and a restriction enzyme.

The nicked plasmid is denatured by 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 extraction kits.

Features

• A long ssDNA (< 1,500 base or < 3,000 base) can be prepared.
• A long ssDNA has a 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) 

 

Citation

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.

 

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)

주문 정보

Product Name	Size	Cat.#	Storage	Detail
Long ssDNA Preparation Kit  for 1.5kb (LsODN Preparation Kit)	1kit	DS610	-80℃	Contact us
Long ssDNA Preparation Kit  for 3.0kb (LsODN Preparation Kit)	1kit	DS620	-80℃	Contact us

NOTICE : ALL PRODUCTS ARE FOR RESEARCH USE on LY. NOT FOR DIAGNOSTIC USE.

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