Single Worm RT-PCR woes

Glenn White gwhite at
Mon Apr 22 10:47:46 EST 2002


We have been having a terrible time with our attempts at single worm 
RT-PCR.  We've encountered every possible
problem, and now are dealing with the fact that we cannot seem to 
prevent degradation of the mRNA before we
amplify it (even though we have gotten amplication of a cDNA before, 
but only sporatically).

We're following the digestion with Proteinase K for 60 min @ 65 oC, 
followed by a kill step, then add the rxn
mix and amplify.

We keep getting amplification of the genomic fragment (primers span 
an intron).  We're using actin primers
right now.

If any of you have had some similar problems and solved them, I'd be 
most appreciative of hearing from you.

We have to do single worm RT, a total RNA prep from a large 
population of animals will not suit our needs.

As always, if I get enough responses, I will summarize them to the 
entire group.

If you are willing to send me your protocol, I'd be grateful for that too.



Dr. Glenn Edward White
Academic Honors Director
Associate Professor of Biology
Ashland University
Ashland, Ohio 44805
"I love being alive and I will be the best man I possibly can.  I 
will take love wherever
I find it and offer it to everybody who will take it.  Seek knowledge 
from those wiser,
and teach those who wish to learn from me."
       Duane Allman

"Emancipate yourself from mental slavery, none but ourselves can free 
our minds."
       Bob Marley

"Do not seek to follow in the footsteps of the wise. Seek what they sought."


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