Reverse Transcription of mRNA

Refer to the RT mastermix calculator for easy adjustment of protocol
 

Master mix for RT	20ml	10ml	5ml RT Reaction Volume
Oligo d(T)16	1ml/tube	0.5ml/tube	0.25ml/tube
RNA:ase inhibitor	1ml/tube	0.5ml/tube	0.25ml/tube
10XPCR Buffer II	2ml/tube	1ml/tube	0.5ml/tube
25mM MgCl2(aq)	4ml/tube	2ml/tube	1ml/tube
dNTP mix	8ml/tube	4ml/tube	2ml/tube
Enzyme
MuLV-RT	1ml/tube	0,5ml/tube	0,25ml/tube
RNA-template standard amount of RNA is 25ng
pAW109/sample	3ml/tube	1.5ml/tube	0.75ml/tube

If MuLV or RNA template have been omitted add DEPC-H₂O to compensate.

RT-reaction (approximate)

1. 15 minutes at 42°C for reverse transcription
2. 5 minutes at 99°C for denaturing
3. 5 minutes at 5°C for cooling

• Finish the RT-mastermix for as many tubes as you need. Adding an extra one will make sure that you don’t come up short on the last tube.
• Distribute 20/10/5ml RT-mastermix into PCR-tubes for all your samples. 
• Allow all tubes to incubate 10 minutes, allowing oligo d(T)₁₆ extension by the RT-enzyme. Extended primers will remain annealed to the RNA template upon raising the reaction temperature to 42°C.
• Perform RT

PCR-cycling (approximate)

1. 12 minutes at 95°C for Ampli-Taq Gold activation, followed by a chosen number of cycles consisting of
2. 15 seconds at 95°C for melting and
3. 30 seconds at 60°C for annealing and extension. The last cycle terminates in
4. 7 minutes at 72°C for final extension and 
5. an optional cold storage at 4°C.

• Prepare the PCR-mastermix for each primer pair (include primers).
• Add the PCR-mastermix to the RT-reaction tubes.
• The reaction volume for each PCR-tube should be 100/50/25ml.
• Perform the PCR.