• 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


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

Protocol used
  • interactome/cdnasyn.txt
  • Last modified: 2019/11/22 17:59
  • by dan