DNA 정제 없이 곧바로 Real Time PCR 적용
Real Time qPCR Master Mix-Terra qPCR Direct TB Green™ Premix

Terra qPCR Direct TB Green™ Premix는 2X master mix로 마우스 꼬리나 귀 조직, 또는 식물의 잎 조직을 정제 없이 crude extract 형태로 Real Time PCR (qPCR)에 곧바로 적용할 수 있다.

qPCR에 사용되는 Terra PCR Direct Polymerase는 whole cell, crude cell lysate 또는 tissue extract그리고 정제된 DNA에서도 뛰어난 증폭 효율을 보이도록 최적화된 non-Taq polymerase로 premix 형태로 제공 된다.
기본이 되는 Terra Polymerase는 GC 함량이 70% 이상인 유전자 적용 시에도 뛰어난 증폭 효율을 나타냄을 확인 하였다(Figure 2). 본 premix 시약은 TB Green™ Green I dye 뿐만 아니라 monoclonal antibody를 사용하여 hotstart PCR을 진행하기 때문에 10분간의 predeanture time 없이도 automatic hot start 반응이 가능하다.

Terra qPCR Direct TB Green™ Premix는 fluorescence signal을 normalization하는데 이용되는 2종류의 ROX reference Dye를 포함하고 있다.

ROX Reference Dye LSR
- ROX Reference Dye LSR는 excitation source가 488 nm laser인 기기에 적합하다.

ROX Reference Dye LMP
- ROX Reference Dye LMP는 excitation source가 lamp 또는 LED인 기기에 적합하다.

ROX Reference Dye 첨가 불필요
Takara사의 Thermal Cycler Dice Real Time System II 또는 Cepheid 사의 Smart Cycler System, Roche사의 LightCycler에는 별도로 첨가할 필요가 없다.

 그림 1

Figure 1. Real-time PCR with crude extracts-Terra qPCR Direct TB Green™ Premix versus a conventional 2X qPCR premix. Real-time PCR was performed using undiluted, 4X diluted, and 16X diluted crude alkaline-heat extracts of mouse spleen or cow muscle (beef foodstuff), and either Terra qPCR Direct TB Green™ Premix or a conventional qPCR premix. Using the manufacturer's recommended conditions for each enzyme mix, a 165 bp region of the β-globin gene Hbb-b1 was amplified from the mouse spleen extract (Panel A), and a 289 bp region of the cytochrome c oxidase gene (COX1) was amplified from the beef extract ( Panel B). Data generated by Terra qPCR Direct TB Green™ Premix corresponded to the theoretical quantities of each gene, while the conventional product was clearly affected by inhibitors present in the crude samples.

 그림 2

Figure 2. Real-time PCR of GC-rich targets-Terra qPCR Direct TB Green™ Premix versus conventional 2X qPCR premixes. Targets with GC-content greater than 70% were amplified by real-time PCR using either human testis cDNA (equivalent to 50 ng-5 pg of total RNA; Panel A) or human genomic DNA (100 ng-10 pg; Panel B) as template, and either Terra qPCR Direct TB Green™ Premix (triangles) or one of two conventional TB Green™ premixes (one specifi­cally for GC-rich targets; see figure legend). Using the manufacturer's recommended conditions for each enzyme mix, gene-specific primers were used to amplify portions of the jun-D proto-oncogene (JUND), the BTB domain-containing protein 6 gene (BTBD6), and the cyclin I gene (CCNI) from the cDNA (Panel A); and a portion of the β-actin CpG island (ACTB_CpG) from the genomic DNA (Panel B). The resulting Ct values were plotted against the initial quantity of DNA used in each assay. Terra qPCR Direct TB Green™ Premix was the only premix able to consistently amplify all of the targets assayed.
Applications
Perform direct real-time PCR from crude extracts, and dirty samples without having to purify template.
Components
Storage Conditions
Store all components at -80℃ after thawing, store the kit-protected from light at 4℃ and use within 6 months.
Do not refreeze!