cDNA preparation protocol – 19/10/2021 – KMG

1) Measure the concentration of isolated RNA with NanoDrop (remember to set RNA as a
measuremen!)
2) Calculate how many μl of water and RNA you need for 50 ng (minimum) – the total volume of
RNA+water cannot exceed 13 μl (look below)
3) Calculate master mix

Component Volume/sample
10x buffer 2 μl
dNTPs (5 mM) 2 μl
Random nonamers (primers) (not in kit) 1 μl
RNaseI (not in kit) 1 μl
Reverse transcriptase 1 μl
RNA + water 13 μl
Tv = 20 μl
All the steps should be done on ice!
4) Prepare the master mix for n+1 of samples (do not warm up the reverse transcriptase stock –
always keep on ice or even better on the special, frozen rack, put back to the freezer asap)
5) Pipet to the labelled tubes H20
6) Pipet the 7 μl of master mix
7) Add RNA
8) Transfer the samples from ice to the cycler (switch it on and find the correct program before
moving the samples)
9) Program: 2h @37C  heat-inactivation 15 or 20’ @65C  cooling to 8C
10) Voila 

Additional remarks:

RNA we keep in -80C, cDNA is more stable, we store in -20C