Long-amplicon QPCR of mtDNA
Posted 07 February 2011 - 06:31 AM
I optimized conditions for the long amplicon (~ 10 kb) and I already got good results which means I got the right product on agarose gel and with 50% reduction of template DNA I got 50% reduction of amplification signal. Unfortunatelly it was not repeatable for the next time and for large amount of samples. Then I started to use AmpliWax Hot start and Genomic-tips for isolation of DNA (instead of Dneasy Kit I used before) because it should isolate high molecular weight DNA and improve the results of long PCR but I got no product in my PCR with this DNA. Does anybody have experience with this type of PCR? I will be happy for any idea!
Thank you in advance.
Posted 07 February 2011 - 11:09 PM
There could be several reasons. The first that comes to mind is inhibition because you use too much DNA. Have you tried diluting your extract?
Another possibility is if your DNA degraded and you don't have so many intact 10kb templates anymore. I would start with the dilution test and then eventually you could run some of your extract on an agarose gel to see if you have DNA and what size it is.Good luck.
Posted 09 February 2011 - 05:31 AM
Edited by janap, 09 February 2011 - 05:42 AM.
Posted 12 February 2011 - 07:16 PM
Is this gel showing your extract? What size are the small bands?
Posted 14 February 2011 - 01:56 AM
Posted 17 February 2011 - 01:30 AM
Just note: running denatured RNA (in formamide) you can obtain visible bands on gel with EtBr, it's commonly used for testing RNA quality. I don't understand the principle either, but RNA can by visualized with EtBr at certain conditions.
Ethidium bromide intercaltes between the 2 strands of dsDNA so I doubt this is RNA. But I don't work with RNA so, I can't guarantee anything.
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.
'Normal' is a dryer setting. - Elizabeth Moon