A collaborator gave me this recipe used for light cyclers. I tried to use it for the Applied StepOnePlus machine and it would not work:

MASTER MIX Q-PCR SYBR Green. (Light Cycler Roche , iCycler BioRad, Stratagene device...)

W1 (polyoxyethylene ether W1 sigma P7516) 0.23%

BSA crystallized (sigma A4378) 0.5 mg/ml

KCl 50 mM

MgCl2 30 mM

dNTPs 0.3 mM each

Glycerol 16.2 %

Platinium Taq DNA pol (invitrogen 10966-034) 0.4 u/ul

AMPD (2 amino 2 methyl-1,3 propanediol sigma A9074) pH 8.5, 0.4 M

SYBR Green I (BMA BioWhittaker 505130) 0.3 - 0.45 % depending batch.

eg :

for 24 ml:

4.48 ml W1 1% in H2O

1.20 ml BSA 10 mg/ml in W1 1% (filtre 0.2 um)

1.20 ml KCl 1M

0.72 ml MgCl2 1M

0.072 ml each dNTP 100 mM

4.48 ml Glycerol 87 % wt/v in H2O Very important : fresh stock

1.92 ml Taq platinium 5u/ul

9.712 ml AMPD 1M pH 8.5

8-10 ul SYBR Green I. Check the color vs an older batch

Aliquot / 0.5 ml

-80 C overnight (very important)

stock long term: –20 C (stable several years)

stock during use: +4 C (stable several months)

Notes :

W1 is a discontinued product.

W1 1% = Brij 56 (sigma P5759) 0.36% + Brij 58 (sigma P5884) 0.64%

100 ml AMPD 1 M pH 8.5: 10,51g AMPD + 84,91 ml H2O + 5.61 ml HCl 38%