Thermoscript RT

Sample OD RNA calculation (1.5ug) RNA amt Add dNTP mix Oligo dT DEPC water Total
name x ug/ul 1.5ug/x = y y ul 2 ul 1 ul z ul 12

Denature the mix by incubating at 65°C for 5 min and then place on ice

Vortex the 5X cDNA Synthesis buffer for 5 s just prior to use

Prepare master mix on ice and vortex gently

1x
5X cDNA Synthesis buffer 4 ul
0.1M DTT 1 ul
RNaseOUT (40U/ul) 1 ul
DEPC water 1 ul
Thermoscript RT 1 ul
Total 8ul

Pipet 8ul of master mix into each reaction tube on ice

Incubate in the PCR machine as the following :

  1. If use Oligo(dT) as the primer = 60 min at 50°C
  2. If use Random Hexamer as the primer = 25°C for 10 min followed by 50 min at 50°C

Terminate the reaction by incubating at 85°C for 5 min

Store the reaction at -20°C or used for PCR immediately

PCR

1X
10X PCR buffer 2.5
50mM MgCl2 0.75
10mM dNTP mix 0.75
Primer Mix (Actin) 1
Taq DNA Polymerase 0.2
dd H2O 18
Total 23

Run in the PCR machine for 30 cycle

Run in the 1% gel and take picture

Save the picture in computer