Jose de las Heras
(by josenet At tiscali.co.uk)
Wed Jan 17 17:11:32 EST 2007
"anton" <noplok At jskhkjgvdskj.kj> wrote in message
news:45ae4bd1$0$49209$14726298 At news.sunsite.dk...
> "Jose McNach" <josenet At tiscali.co.uk> skrev i en meddelelse
> news:1168972999.949719.301750 At v45g2000cwv.googlegroups.com...
>> I normally synthesise my cDNA from RNA using Invitrogen's SuperscriptII
>> reverse transcriptase, and when done I store it at -20oC. I just make
>> dilutions from that stock for semiquantitative PCR and it generally
>> works well, and I can go back to the stocks months later and it's still
>> Last November (2 months ago) I prepared a batch of cDNAs, calibrated
>> them (the dilutions), did a bunch of PCRs, and this time I stored it at
>> -80, together with the RNA. No particular reason.
>> A month later I went back to use some of it, and failed. Then Xmas came
>> and I didn't try again till today... yep, it definitely does not work
>> I think the fact I stored them at -80 and failed is a coincidence...
>> but I *never* have had this sort of problem before so I'm trying to be
>> open minded...
>> Has anybody experienced any problems with cDNA storage at -80?
>> I wonder if freeze-thawing is more harmful at -80 than -20... yet I
>> haven't experienced any problems at all with several rounds of
>> freeze-thawing at -20 in the past...
> We store ALL our cDNA samples at -80, on our hands it is more stable here
> compared to minus 20!
That's what I would have thought too. And I would imagine it being rather
stable, in ds form with RNA (hybrid)... Most people have to be careful with
their RNA and my RNA is just fine!
Someone else observed similar effects (nothing to do with storage
temperature) where cDNAs just stop to work, but they would perform again if
the initial denaturation during PCR is done at higher temperature and a
little longer, which suggests folding issues... So I'm going to try that,
and maybe some DMSO... watch this space.
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