Anybody has a good recipe for homemade SYBR green mix for real time PCR?
To my understanding, one of the key factors is the taq enzyme because most enzymes are inhibited by the SYBR dye.
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%
Is the recipe by Mark working? Please share the experience. Note section is little confusing. Can you please elaborate it @Mark
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