[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.
