The StartScript II cDNA First Strand Synthesis Kit contains two optimized mixes: StartScript II Enzyme Mix and StartScript II Reaction Mix. The enzyme mixture contains StartScript II reverse transcriptase and mouse RNase inhibitor; the reaction mixture contains dNTP and optimized buffer. StartScript II reverse transcriptase is a recombinant M-MLV reverse transcriptase that reduces RNase H activity and improves thermal stability. StartScript II reverse transcriptase synthesizes first-strand cDNA at higher temperatures compared to wild-type M-MLV reverse transcriptase. The enzyme has an activity temperature of up to 48°C and has the characteristics of high specificity and high cDNA yield.
The kit also offers two optimized reverse transcription primers with nuclease-free water contaminated water. The Oligo-dT primer [d(T) 23 VN] can anneale with the start of the polyA tail. The optimized random primer mix can be randomly and continuously paired with the entire RNA template, including mRNA and polyA-free RNA. The synthesized first-strand cDNA product can be up to 10 kb in length.
组分 | UA070076-50 Rxns | UA070076-250 Rxns |
StartScript II Reaction Mix (2X) | 600 μl | 5 x 600 μl |
StartScript II Enzyme Mix (10X) | 100 μl | 5 x 100 μl |
Random Primer Mix (60 μM)
| 110 μl | 5 x110 μl |
Oligo(dT)23 VN* (50 μM) | 110 μl | 5 x 110 μl |
RNase Free dH2O | 1 ml | 5 x 1 ml |
* V = A, G or C; N = A, G, C or T
If denaturation of template RNA is desired, use the following protocol.
1. Mix RNA sample and primer in a sterile RNase-free microfuge tube.
Number | Component | Volume |
1. Template RNA (One of the three is optional) | Total RNA Or poly(A) RNA/mRNA Or specific RNA | < 5 μg < 1 μg < 0.5 μg |
2. Primers (One of the three is optional) | Oligo(dT) 23 VN (50 μM) Or Random Primer Mix (60 μM) Or Specific Primer (1 μM) | 2 µl |
3. H2O | RNase Free dH2O | Up to 8 μl |
2. Denature sample RNA/primer for 5 minutes at 65°C. Spin briefly and put promptly on ice.
3. Add the following components
StartScript II Reaction Mix (2X) | 10 μl |
StartScript II Enzyme Mix (10X) | 2 μl |
4. Incubate the 20 μl cDNA synthesis reaction at 42°C for one hour. If Random Primer Mix is used, an incubation step at 25°C for 5 minutes is recommended before the 42°C incubation.
5. Inactivate the enzyme at 80°C for 5 minutes. The cDNA product should be stored at –20°C. In general, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.
1. Use DEPC to dispose of all equipment used in the study, or purchase equipment that has been proved to be nucleic acid-free. Wear gloves during the study and change them frequently to avoid RNA enzyme contamination.
2. Ensure that there is no RNA enzyme contamination in the reagents used.
3. In order to ensure the effective retrotranscriptional reaction, it is necessary to use high-quality RNA templates.
4. Poly (A)+ mRNA does not need to be isolated from total RNA, but using Poly (A)+ mRNA as a template can improve the yield and purity of the final product. The amount of RNA used, the total RNA is less than 5 μg; Poly (A)+ RNA is less than 1 μg; Specific RNA is less than 0.5 μg.
5. The cDNA products should be stored at -20°C.
6. Enzymes should be placed on ice during experimental operation, and stored at -20°C after the experiment.
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