Storage for PCR

Dan Oerther doerther at merle.acns.nwu.edu
Fri Aug 12 16:54:17 EST 1994


In article <CuFn5L.Ft3 at ucdavis.edu>, ez005139 at chip.ucdavis.edu (Dan
McGoldrick) wrote:

> Hello,
> 
>         We are currently sampling zooplanktonic veliger larvae in the field, 
> formalin killing them and transfering them to denatured alcohol for PCR. 
> We are getting some success in amplification of a mtDNA region but would like
> to improve this to 100%. We get 100 % success with purified mtDNA samples, 
> and pro-k digests of fresh single larvae - so we know the PCR is not the 
> source of failures. We are checking the possibility that the samples 
> are not our species, but the working hypothesis is that some degredation of 
> sample may be occuring.
>    Could anyone offer methods on how to easily prepare field samples of 
> marine planktonic larvae so that we can transport them back to the lab, 
> store them for a year or more, and then perform PCR? Does denatured alcohol 
> work as well as non-denatured alcohol (which is difficult to transport)? 
> Our formalin is dilute and quickly rinsed , but perhaps formalin is the 
> problem? Is there degredation with long term storage at -20 ?
> Any advice will be greatly appreciated...
> 
>                                         Thanx 
> 
> IMC
> Bodega Marine Lab
> P.O. Box 247
> Bodega Bay, Ca.
> 94923

Dan,

   At a departmental seminar recently, a gentleman from Australia was
discussing conducting PCR on waste/drinking water samples.  He discussed
the fact that samples collected from the environment have a greater
tendency to contain contaminants which inhibit PCR.  Therefore, it may be
that your sample is not degraded nor incorrect, but rather, your PCR may
not be working.  One possible way around this is to include an internal
control in each reaction (i.e. some template DNA which should always give
a characteristic band if there are no PCR inhibitors present).  Hope this
helps.

Dan Oerther
Northwestern University



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