SYBR green PCR

SYBR green PCR

PCR master mix
Use the mastermix calculator for calculating adjustments of the protocol

Master mix for PCR	25µl PCR reaction volume
Ampli-Taq Gold DNA-polymerase	0.25µl/tube
25mM MgCl₂(aq)	2.0µl/tube (+2mM)
10XPCR Buffer II	2.3µl/tube (1.5mM MgCl₂)
dd H₂O	16.95µl/tube
SYBR-green 3.75X / 5X (Sigma)	0.5µl/tube
dNTP (mix)	0.5µl/tube (200nM)
Template cDNA	2.0µl/tube (from RT-reaction)

Primers
Add upstream primer	0.25µl/tube end conc 200nM / 1.0µl/tube (20µM) end conc 800nM
Add downstream primer	0.25µl/tube end conc 200nM / 1.0µl/tube (20µM) end conc 800nM

PCR cycle

10 minutes at 95°C for Ampli-Taq Gold activation, followed by a chosen number of cycles:
15 seconds at 95°C for melting and
60 seconds at 60°C for annealing and extension.
Cold storage at 4°C.

To Do

Prepare the PCR-mastermix for each primer pair (include primers)
Add template cDNA to tubes separately or to mastermix if same template in many tubes
Add the PCR-mastermix to the reaction tubes
Spin down reactants
Perform the PCR

Erik Edström, 20010925; latest update 20020529

Reference links: SYBR-green from MolecularProbes Inc; Sigma # S9430