Streamlined method: genome editing 이후, 다양한 Indel sequence 확인
CRISPR/Cas9 뿐만 아니라 TALENs, ZFNs 이후 Indel identification에 사용 가능
Ultrafast protocol: Cell direct PCR과 In-Fusion Cloning을 조합한 빠른 프로토콜
Complete kit: target 의 증폭, 복제, sequencing용 샘플 제작에 필요한 모든 시약 포함
제품설명
Guide-it Indel Identification Kit는 genome editing (CRISPR/Cas9, TALENs, ZFNs) 이후
얻어진Indel (Insertions and deletions)의 다양성을 확인하는 데 최적화된 제품이다. 본 제품은 DNA 서열 분석을
위한 target site의 amplify, clone, sequencing용 샘플 조제를 위한 모든 제품과 프로토콜을 구비하고 있어
신속하고 신뢰성 높은 결과를 얻을 수 있다. Terra PCR Direct polymerase를 사용하므로 DNA 추출, 정제 없이
crude cell lysate로부터 genomic DNA fragment를 증폭할 수 있으며, 증폭산물은 In-Fusion Cloning 방법을 이용하여
pre-linearized pUC19에 바로 cloning 할 수 있다. 이후 제품 내에 포함된 Stellar Competent Cell에 transformation
이후 생성된 Colony는 colony PCR 하여 target region을 증폭한 후 시퀀싱하여 Indel 서열을 분석할 수 있다
(그림 1, 그림 2).
그림 1. The Guide-it Indel Identification Kit provides a complete workflow for identifying the variety
of insertions and deletions (indels) introduced by nuclease-based genome editing. The protocol uses
direct PCR to amplify a genomic DNA fragment (~500 to 700 bp) containing the target site directly from crude cell
lysates (step 1). The resulting amplified fragments contain a pool of edited target sites from individual cells.
These PCR products are cloned directly into a pre-linearized pUC19 vector using the In-Fusion cloning system (step 2).
After transformation of an optimized E. coli strain, colony PCR is used to amplify the target site from
the plasmid (step 3). DNA sequencing is then used to identify the different indels generated at the targeted genomic
site (step 4).
그림 2. Identification of insertions and deletions (indels) in the CD81 gene after CRISPR/Cas9 targeting.
HeLa cells were transfected with plasmids encoding Cas9 and an sgRNA targeting the CD81 gene. The Guide-it Indel
Identification Kit was used to prepare CD81 target sites for DNA sequence analysis. The sequencing data from six clones
was aligned with the wild-type sequence, revealing a broad range of indels in the CD81 gene.
적용
Detecting insertions and deletions introduced in mammalian genomic DNA
Characterizing the variety of indels that are present in a cell population after nuclease-based gene editing
구성품 (자세한 내용은 CoA를 참조하세요)
Guide-it Indel Identification Components
110 μl Terra PCR Direct Polymerase Mix
3 x 1 ml 2X Terra PCR Direct Buffer (with Mg2+, dNTP)
