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-   -   syber green master mix home made (http://www.molecularstation.com/forum/real-time-pcr-quantitative-pcr-forum/82899-syber-green-master-mix-home-made.html)

wenmin 06-02-2011 05:12 PM

syber green master mix home made
 
i have made syber green master mix by Duncan Clark's recipe. The amplification curve looks good. However the dissociation curve was wired. The recipe is

Stock reagent Volume(10mL) Final
1M Tris pH 8.3 1mL 100mM (UV irradiated)
25mM MgCL2 (Qiagen) 2.4mL 6mM
10mg/mL BSA Omnipure Fraction V 1mL 1mg/mL
10mM dNTP Mix 400μL 400μM
100X SyBR Green 66.67uL 0.66X
dH2O (irradiated DEPC) 5.032 mL

Does anyone can give me suggestion about the wired dissociation curve?

sunglassesatnight 08-03-2011 11:35 PM

Re: syber green master mix home made
 
Is this a 10X master mix? If so it looks probably OK, although I don't know why you would add BSA. And there is no source of monovalent cations. I would guess that the weirdness in your melt curves comes from your primers (unless they give normal peaks with other buffers) or from the lack of a passive reference dye. Some machines (ABI does) need a passive signal to generate a normal looking melt curve. My master mix (also home-brew) as a 10X stock is this:
100mM Tris-HCl (pH8.3)
500mM KCl
30mM MgCl2
0.1% Tween 20
8% Glycerol
1:4000 SYBR Green I
1:36 ROX reference dye
It works really well for me and gives excellent amplification and melt curves.

butters 09-28-2011 10:52 AM

Re: syber green master mix home made
 
Thank you wenmin and sunglassesatnight for sharing, I would like to know have you solved the problem wenmin?

if still persist, can you run a realtime reaction before storage on low temperature? It would be awesome if you could show us how "weird" is the melt curve. Thanks.

wenmin 09-29-2011 12:29 PM

Re: syber green master mix home made
 
yes, we solve this problem by adding rox reference dye into the master mix. Thanks for the suggestion from Sunglassesatnight.


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