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How fast do DH5-alpha cells with plasmid insert lyse after defreezing? - (Jul/18/2009 )

I had taken out DH5-alpha cells with plasmid inserts from our lab's frozen stocks to streak on petri dishes. However, I had stuck them only in ice for ~30 mins and afterwards, the frozen stocks with glycerol had defrosted. I then stuck the now-liquified stocks into dry ice to refreeze before streaking on petri dishes. Today when I checked my petri dishes, only one of the stocks had grown. I am wondering how fast DH5-alpha cells lyse and if this is the reason for no bacterial growth.



Hi Princesslinda -- welcome to the BioForums!

Though not the greatest practice, I have on occasion completely defrosted frozen glycerol stocks of plasmid-containing DH5alpha, and struck some of the liquid out onto plates. The liquefied stocks were placed back in the -80C freezer.

The plates grew fine, and the stock was also fine on subsequent use.

So, I don't think defrosting glycerol-protected stocks causes significant lysis...