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syber green master mix home made

syber green master mix home made - Real-Time PCR and Quantitative PCR Forum

syber green master mix home made - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 06-02-2011, 05:12 PM
Pipette Filler
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Default 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?
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Old 08-03-2011, 11:35 PM
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Default 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.
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wenmin (08-08-2011)
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Old 09-28-2011, 10:52 AM
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Default 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.
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Old 09-29-2011, 12:29 PM
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Default 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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