I run a native 12%PAGE (1X TBE buffer + 20 mM Mg2+ as running buffer), and the molecular beacon was labeled on 5' and 3' with FAM and BHQ1, respectively. Running condition is 80 V, 80 min in an ice bath. After I stained in SYBR gold for 30 min, and I only see the hybrid dsDNA band with unlabeled DNA (only lane 4 and 5). Does anyone have any clue about such an issue?
From the left to right lanes:
1. DNA ladder
2. 1 µM molecular beacon (labeled on 5' and 3' with FAM and BHQ1, respectively.)
3. 0.2 µM molecular beacon (labeled on 5' and 3' with FAM and BHQ1, respectively.)
4. 2 µM complementary DNA + 1 µM molecular beacon (labeled on 5' and 3' with FAM and BHQ1, respectively.)
5. 2 µM complementary DNA
Thank you very much.
Looking forward to hearing from you,
Sin-Cih
Since your gel conditions are not denaturing, the molecular beacon is presumably in its self-quenched secondary structure. So the FAM on the MB probably is not contributing significant fluorescence. The BHQ may also be quenching the fluorescence of any SYBR Gold that is binding to the MB. Nevertheless, there is a faint band in lane 2, between the lowest 2 ladder bands.
Adam B Shapiro Thank you for your reply. I agree with your point due to the BHQ1 quenching the fluorescence. But I am confused that molecular beacon can make it “off-on” because of FRET between FAM and BHQ1 in a very close distance. I remember SYBR gold is a groove-binding unsymmetrical cyanine dye for staining DNA, so propose it can binding whole DNA strand and the FRET distance of SYBR gold may far from the BHQ1. (over 10 nm the FRET efficiency is very low)
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