Quantitative RT-PCR


Using Invitrogen™ kit: Superscript™ III Platinum® SYBR® Green One-Step qRT-PCR (ref #11736-051)

Setup your cycling program and your plate annotation on a Stratagene MX3000P machine.
Prepare, on ice, in "RNA-world" area with gloves and RNA-dedicated labware, for n reactions :

Master Mix :
 H2O :
 n x 8.68 ul
 Primer mix (5 uM each)
 n x 0.88 ul
 2X SYBR Green Reaction Mix :  n x 11 ul
 Superscript III RT/Platinum Taq Mix :  n x 0.44 ul

Dispatch 21 ul of this mix in each reaction well.
Add 1 ul of RNA template in each reaction well.

A typical cycling program :

50°C for 3 minutes
95°C for 5 minutes
40 cycles of :
    94°C for 15sec
    51°C for 30sec with Acquisition at endpoint.
    72°C for 15sec
95°C for 1 minute
51°C for 30sec
95°C for 30sec with Acquisition during ramp.

Dernière mise à jour : ( 04-03-2009 )