Case of the disappearing cDNA - (Aug/01/2007 )
cDNA was synthesized from tRNA (Trizol method). The cDNA kit used was from Fermentas (and i used random hexamers).
I used the cDNA for a PCR reaction right after it was synthesized, and got a nice band of expected size. I froze the cDNA in -20C, and the next day, had to use it to amplify another transcript... and got no product. On the third day, I recheck it with control primers (which worked on day I) and still got no product...
Has anyone experienced this before? How can I keep the cDNA from 'disappearing' so I do not have to re-do cDNA synthesis 'everyday'...??!!
thanks! 
-lurena-
QUOTE (lurena @ Aug 1 2007, 03:50 PM)
cDNA was synthesized from tRNA (Trizol method). The cDNA kit used was from Fermentas (and i used random hexamers).
I used the cDNA for a PCR reaction right after it was synthesized, and got a nice band of expected size. I froze the cDNA in -20C, and the next day, had to use it to amplify another transcript... and got no product. On the third day, I recheck it with control primers (which worked on day I) and still got no product...
Has anyone experienced this before? How can I keep the cDNA from 'disappearing' so I do not have to re-do cDNA synthesis 'everyday'...??!!
thanks!
I used the cDNA for a PCR reaction right after it was synthesized, and got a nice band of expected size. I froze the cDNA in -20C, and the next day, had to use it to amplify another transcript... and got no product. On the third day, I recheck it with control primers (which worked on day I) and still got no product...
Has anyone experienced this before? How can I keep the cDNA from 'disappearing' so I do not have to re-do cDNA synthesis 'everyday'...??!!
thanks!
Well I keep my cDNA's at -20 for much longer than one day. What is different about your PCR? I would guess it to be a PCR related problem before I say the cDNA disappeared.
You should be able to keep it at least 6 months at -20, given that you take care of it- thaw it on ice a couple minutes before you want to use it and briefly microfuge before each use.
Maybe troubleshoot your PCR?
-Vicky.ac-
Is it possible that may be one of the solutions could be contaminated which is degrading the DNA.
-scolix-