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cDNA stability?

cDNA stability? - Protocols and Methods Forum

cDNA stability? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 01-16-2007, 06:43 PM
Jose McNach
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Default cDNA stability?




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
okay.

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
anymore.

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...

hmmm...

Any comments? Thanks!

Jose

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  #2  
Old 01-17-2007, 09:54 AM
StewJW
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Default cDNA stability?

Superscript II is not stable at -80 C which is why it is shipped from
Invitrogen on wet-ice and not dry-ice. Superscript III however is
shipped on dry-ice and is more stable at lower temperatures although
recommended storage for both enzymes is -20 C.
..

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  #3  
Old 01-17-2007, 04:16 PM
anton
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Default cDNA stability?


"Jose McNach" <[Only registered users see links. ].uk> skrev i en meddelelse
news:1168972999.949719.301750@v45g2000cwv.googlegr oups.com...

Hi.
We store ALL our cDNA samples at -80, on our hands it is more stable here
compared to minus 20!


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  #4  
Old 01-17-2007, 10:06 PM
Jose de las Heras
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Default cDNA stability?

I don't store the enzyme at -80, but the cDNA produced with it.

Jose


"StewJW" <[Only registered users see links. ]> wrote in message
news:1169027660.052932.296230@38g2000cwa.googlegro ups.com...
Superscript II is not stable at -80 C which is why it is shipped from
Invitrogen on wet-ice and not dry-ice. Superscript III however is
shipped on dry-ice and is more stable at lower temperatures although
recommended storage for both enzymes is -20 C.
..


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  #5  
Old 01-17-2007, 10:11 PM
Jose de las Heras
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Default cDNA stability?


"anton" <[Only registered users see links. ]> wrote in message
news:45ae4bd1$0$49209$[Only registered users see links. ]...

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.

Jose


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