silver stained TGGE bands: ssDNA vs. dsDNA...

Roland Hübner rhubner at gins.uia.ac.be
Thu Mar 25 05:42:34 EST 1999


Hello bionetters,

 when I load a homogeneous CTRL PCR product (250 bp) in low-salt TBE
buffer for TGGE analysis I obtain two bands rather than the expected
single band... The faster moving band stains blackish, whereas the slower
moving band is orange/
red-brown... I read that this (extra) band corresponds to ssDNA...

 BTW, I tried denat./renat. in the presence of about 4M urea at 5 min
95oC/15 min 50oC vs. 10 min 95oC/20 min 55oC, also with 2O min ramping,
and get same gel pattern... (=> heteroduplex TGGE)... The gels are run
with a gradient from 20oC to 50oC on a PELTIER-based system...

 A perpendicular gradient analysis determined the melting for one product
to start at 30oC, but others claim that this product shows just one single
band with their HD-TGGE protocol...

 Any ideas for explaining this discrepancy?

 Thank you very much for advice - obviously I am new to conformation
analysis (good reading to recommend about the 'principles'?)

Kindest regards,
 Roland

ps. I can send gel picts by email if necessary (albeit black & white "only")



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