Protocol for 1st strand cDNA synthesis
Material
- Sample : Total RNA sample
- PrimeScript™ II 1st strand cDNA Synthesis Kit (TaKara, Cat.6210A)
PrimeScript™ II 1st strand cDNA Synthesis Kit (For 50 reactions)
| Components | Volume | Storage | Memo |
|---|---|---|---|
| PrimeScript II RTase（200 U/μl） | 50 μl | －20°C | |
| 5×PrimeScript II Buffer | 200 μl | －20°C | |
| RNase Inhibitor（40 U/μl） | 25 μl | －20°C | |
| dNTP Mixture (10 mM each) | 50 μl | －20°C | |
| Oligo dT Primer（50 μM） | 50 μl | －20°C | |
| Random 6 mers（50 μM） | 100 μl | －20°C | |
| RNase free dH2O | 1 ml | －20°C |
Protocol
1 Reaction preparation
- Mix the following reagents in one tube
| Reagent | Volume | Note | |
|---|---|---|---|
| Oligo dT Primer（50 μM） | 1 µL | ||
| or Random 6 mers（50 μM） | or 1 µL（0.4～2 µL） | For genes <2kbp, use1～2 μl. When >2 kb, 0.4～1 μl is recommended. | |
| or Gene specific primer (1 µM) | or 1 µL | Final concentration at 0.1 μM. | |
| === | |||
| dNTP Mixture（10 mM each） | 1 µL | ||
| RNA Template | ~8 µL | Total RNA：<5 μg, polyA＋ RNA：<1 μg | |
| RNase Free dH2O | up to 10 µL | ||
- Incubate at 65˚C for 5 mins
- Transfer tube to ice immediately
! IMPORTANT STEP ! By doing this, the RNA template degenerates and the reverse transcription efficiency increases.
2 Reverse transcription
- Add the following reaction mix, and make the total volume 20 µL.
| Reagent | Volume | Note |
|---|---|---|
| Reaction mix from previous step | 10 μL | |
| 5×PrimeScript II Buffer | 4 μL | |
| RNase Inhibitor（40 U/μl） | 0.5 μL（20 U） | |
| PrimeScript II RTase（200 U/μl） | 1 μL（200 U） | |
| RNase Free dH2O | up to 20 μL |
- Mix gently
- Immediately proceed to the following reaction
| Temprature | Time | Note |
|---|---|---|
| 30˚C | 10 min | Only when using random 6mer primers |
| 45˚C | 30-60 min | |
| 75˚C | 15 min | Deactivation. Paramater for long transcrpits just in case. |
| 4˚C | Forever | (Immediately put on ice.) |
- Dilute reaction to 50 μl with 30 μl H2O for PCR.
- The cDNA product should be stored at -20°C.
- For downsteam PCR amplification, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.
Protocol last updated
— Dan Yamamoto-Evans 2019/11/21 15:16