I tried home made syber green master mix which's recipe was posted by Duncan Clark as below.
makes 10mL store in aliquots in the -20
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
**for use: Add 10μL Qiagen HotStarTaq per 1mL Master Mix
We got pretty good amplification curve and very wired dissociation curve with the home made master mix. The wired dissociation curve is not a curve at all. I could not see where is the baseline, where is the main peak. BTW, all the primers have been tested. I run real-time with ABI syber green master mix on the same plate, all the ABI's wells run perfectly.
Does any one has the same problem? Or any suggestion for this issue?
Thanks a lot.