Hi, I received a frozen vector (rAAV) solution and thawed it to aliquot. I was unaware that I had to thaw the solution on wet ice and thawed it by rubbing the bottle in between my hands. Big mistake. Then, I aliquoted half of the vector solution. Here’s the other mistake. My aliquot conical tubes were placed on a tube rack at room temp, rather than on wet ice. The aliquots moved from room temp to a -80°C freezer.
Will my aliquots and original vector solution work like intended or did I just waste my vector solution?
Thank you.
To say you have a "vector solution" is not really helpful. There are many different meanings of vector. I am however presuming that in this case you mean a plasmid DNA that you wish to clone into? If that is the case, then you don't need to worry. DNA is quite stable, especially if uncut. If you have pre-digested plasmid DNA then usually we like to keep it cold to reduce any potential nuclease activity that might nibble at the single stranded overhangs. But again I wouldn't really worry.
However if by vector you mean something else, then ignore my answer entirely.
I wouldn't worry too much. Although ssDNA is more sensitive than dsDNA, temperature is not a problematic condition. Remember that these things are in our bodies at 37C all the time.
Dear Chau Do ,
I agree with Michael J. Benedik . If you mean the AAV construct plasmids then there is nothing to worry about.
However as he rightly pointed that vector could have several meanings. It can also mean the final rAAV particles that are generated from AAV production protocols.
If it would have been the rAAV vector particles that were thawn by hand (which were to be used for transducing target cell or tissue) then you might have some issues for your second set of experiments with the AAV particles that you re-froze.
Thawing by hand "might" have degraded the functional viral particles and the additional re-freezing of remaining AAV particles will lead to freeze thaw damage. Therefore, for the next round of transduction experiment, you might encounter lower/poor transduction efficiency but the virus will work as intended.
To avoid intense freeze-thaw damage, it is advisable to thaw the rAAV particles on ice.
Good luck !
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