Hi,
I conducting one-step real time RT-PCR, I get a single peak of melt curve but 2 products were observed analyzed by agarose gel. I run together with non-RT and non-template and there is no products for both negative control. Can anybody pls help me figure what's the problem? The primers are specific and I having the same problems for few genes that study.
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#2
littleaxt
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Posted 03 March 2009 - 11:58 PM
Hello,
What is the range of your melt curve analysis? Maybe one of your products is already denaturated, because you start with a too high temperature?
All the best!
What is the range of your melt curve analysis? Maybe one of your products is already denaturated, because you start with a too high temperature?
All the best!
chloe, on Mar 4 2009, 02:20 AM, said:
Hi,
I conducting one-step real time RT-PCR, I get a single peak of melt curve but 2 products were observed analyzed by agarose gel. I run together with non-RT and non-template and there is no products for both negative control. Can anybody pls help me figure what's the problem? The primers are specific and I having the same problems for few genes that study.
I conducting one-step real time RT-PCR, I get a single peak of melt curve but 2 products were observed analyzed by agarose gel. I run together with non-RT and non-template and there is no products for both negative control. Can anybody pls help me figure what's the problem? The primers are specific and I having the same problems for few genes that study.
#3
toejam
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Posted 04 March 2009 - 02:27 AM
maybe your gene presents splicing, thus 2 different products. have you sequenced them to see if they're similar or just rubbish?
"When there's no more room in hell the dead will walk the Earth"
#4
chrisbelle
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Posted 06 April 2009 - 01:39 AM
chloe, on Mar 4 2009, 09:20 AM, said:
Hi,
I conducting one-step real time RT-PCR, I get a single peak of melt curve but 2 products were observed analyzed by agarose gel. I run together with non-RT and non-template and there is no products for both negative control. Can anybody pls help me figure what's the problem? The primers are specific and I having the same problems for few genes that study.
I conducting one-step real time RT-PCR, I get a single peak of melt curve but 2 products were observed analyzed by agarose gel. I run together with non-RT and non-template and there is no products for both negative control. Can anybody pls help me figure what's the problem? The primers are specific and I having the same problems for few genes that study.
are your products on the gel of the same size? are both your expected product size? did you repeat your run to see if that was just a freak run or it happened again? Usually 1 melt curve only begets 1 product size. If your 2nd product on gel is <100bp, highly likely that is primer-dimer.
All the best,
Chris
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