4 replies to this topic

[rajgene]

hi guys,
planning to do pcr cloning of a promoter. will transfect the resulting reporter plasmid in cos cells. if i isolate the vector plasmid DNA from a megaprep. how long i can store it in the freezer. does long term storage cause any deterioration of the plasmid DNA.
Also please let me know the storage conditions.
care
rajgene
THX

[green]

I think you can store plasmid DNA at -20C forever. I have used 5-years old plasmid such stored.

Edited by green, 06 January 2005 - 12:40 PM.

[DointheScience]

At -80C, the plasmid should be stable in water for years.  Be sure and aliquot to avoid multiple freeze-thaw cycles.  Avoid self-defrosting freezers.  I don't know if that matters, but I always hear it.

Over-time, plasmids begin to deteriorate, but probaly not enough to prevent their use.  If using old plasmids and you have trouble, you can run some on a gel (0.8-1.0% agarose), cut the band of supercoiled DNA, and purify to get rid of excess nicked DNA.

In addition to storing the plasmid, store a couple 1 ml aliquots of the transformed bacteria to innoculate culutres down the road.  Add 300ul 50% glycerol to 700ul of your maxiprep culture and freeze.  Try to use microtubes that have a rubber sealing ring in the lid.  These bacterial stocks should be safe for years as well.


- The Biology Ninja

Edited by DointheScience, 08 January 2005 - 01:24 PM.

[jadefalcon]

dna should be stable at low temperatures for long times. i used plasmids stored for >7 years in a self-defrosting -20°C freezer. if they are no more ready to use "out of the box", i just transform them into fresh competent cells and there you go - new plasmid, all ok. and BTW i once forgot to freeze some minipreps for a longer period, 6 months that is, and left them in the cooler at 4°C. were just fine after half a year! could have beep just plain luck, though.

mike

[DevGrp]

The problem with self defrosting freezers is that they cycle their temperature (heating up cooling down ) to avoid frosting up. This can have the effect of continually defrosting / refreezing your samples. They can also dry out samples in a similar way to freeze drying