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-20 freezer fail =(. Help, please!!!

antibiotics polymerases IPTG T4 ligase

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6 replies to this topic

#1 Altair

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Posted 16 October 2011 - 05:28 PM

Hi all,

I went into lab last Friday morning to find that sometime Thursday night (presumably after 6pm or so), our -20C freezer stopped working. Consequently, a bunch of my aliquoted chemicals/PCR supplies were sitting at 4C or higher for up to ~ 12 hours (everything was thawed and there was a huge puddle of water under the freezer). So I'm trying to decide which aliquots are still good and which I should throw out. I'm fairly sure my plasmid stocks/PCR primers are okay, but everything else is up in the air. Would appreciate your input on whether the following items could survive 12 hours at > 4C:
  • antibiotic stock solns (ampicillin in water, kanamycin in water, chloramphenicol in ethanol, tetracycline in ethanol)
  • Pfu and Vent polymerases and their buffers
  • dNTPs
  • T4 DNA ligase and T4 ligase buffer
  • IPTG (1M in water)
  • actinomycin D stock soln
  • MTT stock soln
  • Laemmli sample buffer (with and without DTT)
  • Dpn I restriction enzyme
I'd really rather not throw out anything more than I have to, but I'd also rather not risk having stuff fail because of bad reagents. Any insight you guys could give would be great!


Thanks!!

#2 phage434

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Posted 16 October 2011 - 11:18 PM

I would toss the dNTPs and the T4 ligase and its buffer. I think the rest is probably ok. I don't know anything about the actinomycin and MTT.

#3 Shrubal

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Posted 17 October 2011 - 07:18 AM

MTT is stable for almost a month at 4oC. antibiotics are also good for 2months at least at 4oc

#4 bob1

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Posted 17 October 2011 - 02:33 PM

The laemmli buffer without DTT will be fine, if the stuff with DTT has gone red/brown then throw it out. I (personally) would throw out any enzymes - it's usually not worth the risk of having something fail and trying to find out why (could be the enzyme, but it might not be), when you can just get some more in, which you will have to do anyway to test if it is the enzyme that has failed.

#5 Altair

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Posted 17 October 2011 - 06:36 PM

Thanks for all the replies so far! Hm, apparently the Laemmli buffer with DTT is fine (ran a reducing SDS PAGE gel today). I've tossed the T4 ligase and its buffer (there was some weird precipitate at the bottom) and gotten new dNTPs. Won't know about the MTT until later (but good to know it's stable at 4C for that long). Going to test the Vent and Pfu polymerases tomorrow against new stock...

Turns out today some of my freezer boxes got left on top of the freezer at room temp because someone took them out (presumably to reach their stuff) and forgot to put them back in. It was long enough for my 1-ml IPTG aliquots to thaw, but not longer than an hour I think. At least the box with my enzymes and stuff wasn't one of the ones left out. *sigh*.

#6 bob1

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Posted 18 October 2011 - 05:21 PM

The ppt in the ligase buffer is quite common. I think it is the DTT in the buffer that precipitates. If you read the instructions for the T4 it tells you to vortex the buffer to resuspend the ppt.

#7 Altair

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Posted 19 October 2011 - 08:14 AM

Oh, all right, thanks for the insight! I'll keep that in mind next time.

According to test PCRs, my Vent polymerase at least is fine, so am hoping the Pfu's also okay.





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