The StartScript cDNA First Strand Synthesis Kit contains two optimized mixes: M-MLV enzyme mix and M-MLV reaction mix. The M-MLV enzyme mixture contained M-MLV reverse transcriptase and mouse RNase inhibitor, and the M-MLV reaction mixture contained dNTP and optimized buffer. The kit also contains two optimized reverse transcription primers and nuclease-free water. The anchored Oligo-dT primer [d(T) 23 VN] forces the primer to anneale the start end 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 longer than 10 kb in length.
组分 | UA070075-50 Rxns | UA070075-250 Rxns |
M-MLV Reaction Mix (2X) | 600 μl | 5 x 600 μl |
M-MLV Enzyme Mix (10X) | 100 μl | 5 x 100 μl |
Random Primer Mix (60 μM)
| 110 μl | 5 x 110 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
1. Denatured RNA template
The following mixture is prepared in the RNase-free centrifuge 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 |
The RNA is denatured for 5 minutes at 70°C, centrifuged briefly, and immediately placed in ice. This step is optional. However, it can increase the cDNA production of long messenger Rnas and GC-rich RNA regions.
2. Prepare the first strand cDNA synthesis reaction solution and gently blow and mix with a pipette.
3. The first strand cDNA synthesis reaction was carried out according to the following conditions
The 20 μl cDNA synthesis reaction was incubated at 42℃ for 1 hour. If using a random primer mixture, it is recommended to incubate at 25°C for 5 minutes before incubation at 42°C. Inactivate the enzyme for 5 minutes at 80°C. The cDNA product should be stored at -20°C. In general, the volume of the cDNA product should not exceed 1/10 of the volume of the PCR reaction.
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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