How to synthesis cdna without using kit?

1. Extract RNA
2. Quantify its value (upto1 µg:1 ng-1 µg total RNA or pg- ng poly (A)-RNA)
3. Put on ice immediately after incubation
4. Add dNTPs (mM), nuclease-free water and the total volume should be µL. Primer concentration should be optimized according the primer type and initial amount of RNA.
5. Centrifuge it and work on ice
6. Add enzyme reverse transcriptase, its buffer and RNase Inhibitor. Volume should be adjusted with nuclease-free water in µL.
7. Temperature and incubation time depends on type of enzyme reverse transcriptase (AMV, M-MuLV) 
8. Inactivate the enzyme at°C for5 minutes.
9. After cDNA synthesis store the tubes at -ºC.

on utilise des kittes d'extraction et de purification d'ADN afin d'ameleorer la technique d'amplification par PCR et ainsi southen blott 

In the RNA sample (up to1 µg :1 ng-1 µg total RNA or pg- ng poly(A)-RNA) add primer oligo d(T) or  Random Primer(octamer or decamer). Incubate5 min at (for oligo dT) or min at°C for random primer. Immediately put on ice after incubation.

Add dNTPs (mM), nuclease-free H2O . Total volume shoud beµL. (concentration of primers should be optimized according the initial RNA amount and primer type)

Spin briefly, work on ice...

Add reverse transcriptase, its buffer and RNase Inhibitor. Adjust volume toµL with nuclease-free H2O.

Incubation time and temperature depends on type of reverse transcriptase  (AMV, M-MuLV) 

Inactivate the enzyme at°C for5 minutes. The cDNA product should be stored at -°C.

Consider if you may need reverse transcriptase with or without RNase H activity. 

steps for cDNA synthesis:

1- isolate RNA

2- quantify its value for further reaction

3- prepare a reaction  containing;  oligo dT, water, RNA ample.

4- give it a heat shock at 60 degree for 5 min. and then place on ice immediately.

5- then add 5X reaction buffer, dNTPs, ribolase inhibitor and MMLV-RT.

6- place the tube in thermocycler  at specified temperatures.

7- after cDNA  synthesis stores tubes at -20.

By getting the random hexamer or oligo dt primers synthesized and setting up the PCR reaction using reverse transcriptase enzyme.