Description/Introduction
This product contains 5× Ready-to-Use Super Master Mix (reverse transcriptase cocktails including Reverse Transcriptase, RNase Inhibitor, Random Primer, Oligo (dT)18 Primer, dNTPs and the optimized buffers), No reverse transcriptase control (has the same compositions as Super Master Mix except the reverse transcriptase enzyme) and one step gDNA remover reagents. It can remove genomic remnants of the template before the reverse transcription reaction is performed. Reverse Transcriptase (RT) is a mutant reverse transcriptase obtained by in vitro evolutionary screening and is characterized by high thermal stability and processivity. gDNA Remover is a thermosensitive DNase that is 30 times more active than DNase I and works only on dsDNA, gDNA Remover is susceptible to inactivation under high temperature conditions.
Storage and Handling Conditions
Shipping with blue ice, stored at -20℃ upon received.
Component
| Component Number | Component | G3337-50 | G3337-100 |
| AG3337 | 5× cDNA Super Mix for qPCR | 200 μl | 400 μL |
| AG3337-R | gDNA Remover | 50 μL | 100 μL |
|
| Nuclease-Free Water | 1 mL | 1 mL |
| G3337-C | 5× No-RT Super Mix Control | 20 μL | 40 μL |
Assay Protocol / Procedures
Synthesis of the first strand cDNA and Genomic DNA removal
(1) Reaction setup for 20 uL volume:
| Component | Volume |
| 5× cDNA Synthesis Super Mix for qPCR | 4 μL |
| gDNA Remover | 1 μL |
| Total RNA/mRNA | 0.1 ng-5ug / 10 pg-0.5ug |
| Nuclease-Free Water | Add to 20 μL |
(2) Gently mix and centrifuge.
(3) Setup reverse transcription program on a PCR machine
|
| Temperature | Time |
| Primer annealing | 25℃ | 5 min |
| cDNA synthesis | 42℃ | 15-30 min |
| Enzyme Inactivation | 85℃ | 5 s |
| Storage | 10oC | varies |