● Complete system for 1st-Strand cDNA synthesis
● Active at 42~55℃. Optimum at 50℃
● Superior performance in RT-PCR.
● cDNA synthesis with high processivity and sensitivity
HelixCript™ 1st-Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from total RNA or poly(A)+-selected RNA. This kit uses HelixCript™ Thermo Reverse Transcriptase, a thermostable and RNaseH negative variant of M-MLV RTase, that is active at a temperature range of 42℃ ~ 55℃. At the escalated temperature, the cDNA synthesis is enhanced partially due to the less internal structural formation of the template RNA and increased polymerization activity of RTase. The included RNase inhibitor in this kit protects the template RNA from degradation in the reaction mix and enhances the yield of cDNA. First strand cDNA synthesized with this kit can be directly used as a template in a downstream assay such as PCR or real-time PCR.
|◎ Generation of first strand cDNA for subsequent PCR or Real-time PCR |
◎ cDNA library construction
50 pmole/㎕ oligo d(T20) primer
50 pmole/㎕ random hexamers
dNTP mix (each 10mM)
5x RT reaction buffer
HelixCript™ Thermo Reverse Transcriptase (200unit/㎕)
HelixCript™ 1st-Strand cDNA Synthesis Kit was functionally tested for RT-PCR amplification on different sized products (0.5 ~ 5 kb) with 500 ng of total RNA. 500 bp RT-PCR product was obtained from 100 pg of total blood RNA using human β-actin primers.
|Figure 1. RT-PCR using HelixCript™ 1st-Strand cDNA Synthesis Kit and random hexamer or oligo-dT as primers for reverse transcription. 1st-strand cDNA synthesis reactions were performed using the 300 ng total RNA isolated from human blood. 10% of each 1st-strand cDNA products were used in PCR reactions with primer sets corresponding to fragment of human dynein gene and 1.25 units HelixAmp™ Xtender DNA polymerase in 50 ul PCR reaction. |