UCSF

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

8 ul

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

Incubate in the PCR machine as the following :

  • If use Oligo(dT) as the primer = 60 min at 50°C
  • 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