Reviving dead glycerol archive - (Aug/03/2005 )

Our lab has several glycerol archive plates that have been subjected to freeze/thaw cycles and/or aging. From these plates we are trying to resequence what is in the plates. Most wells grow and have sequenced fine. However it seems that the most important clones are in the wells that will not grow when replicated for analysis.

I was wondering if anyone has tried to revive a clone from a dead glycerol archive well and if so how?

We have tried PCR and then transforming the product but if we get a product at all where the band is approximately the size desired it does not sequence any where close to the orginial. We have also tried letting the culture grow for 24 hours to see if there would be any growth but there was no luck here either.

Any ideas would be appreciated, even if you havent tried to bring back dead clones.

to get some precious plasmid back, i once successfully did a plasmid extraction directly with the "glycerolised" e.coli, eluted in 30µl and used 20µl of that for chemical transformation of ultracompetent cells. i ended up with two clones only, but they where "the right ones". so, dead stock revived. but if this works all the time or if i've bee plain lucky, i can't tell.

mike

Mike,

well it is worth a try. If it worked once it can work again. Thanks for the suggestion.

Howdy,

I asked a similar question yesterday but for bacterial clones from dried agar plates. I had heard of a mysterious "Lazarus" technique from FOCUS (Invitrogen) newsletter and they kindly faxed me the article yesterday. The method is for dried agar plates (with "agar the consistency of potato chips" ) and agar stabs, but I don't see any reason why it shouldn't work for glycerol stocks.

Eissenberg, J.C. (1993) "The Lazarus technique, or, recombinant molecules back from the dead" FOCUS 15, 53.

For dried agar plates: cut out a section of plate with dried bacterial residue. Place in a microcentrifuge tube containing TE [10mM Tris-HCl (pH 8), 1mM EDTA] and 1% SDS

For agar stabs: add TE plus SDS directly into the stab vial using an appropriate volume. Using pipette tip, break up the agar into small fragments, then decant the contents into a microcentrifuge tube

1) Vortex suspension at high speed for 3-5 min
2) Spin at high speen for 30 sec
3) Remove supernatant liquid to a fresh tube
4) Extract supernatant with an equal volume of phenol:chloroform (1:1). Centrifuge 5 min at high speed and remove aqueous phase
5) Extract with an equal volume of chloroform. Centrifuge as above and remove aqueous phase
6) To the aqueous phase, add one-tenth volume 3M sodium acetate (pH 5.5) and two volumes absolute ethanol
7) Recover precipitated material by centrifugation at high speed for 10min
8) Discard supernatant liquid and dissolve pellet in 10ul sterile water
9) Use entire amount for transformation of bacteria

Good luck! If you'd like the whole article I'm happy to send it on - Drop me an email.

Nicole