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cloneJet (Fermentas) ligation buffer "stability"


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#1 talianka

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Posted 11 March 2013 - 04:05 AM

Dear colleagues,
I would like to ask a question regarding the "stability" of the buffer included in the commercial cloneJet cloning kit (Fermentas). The cloneJet kit does blunt-end ligation, involves the blunting enzyme, so the buffer has to work not only for the ligasae, but for the blunting enzyme too... Today, I asked a student to aliquot the buffer to avoid repeated thawing and freezing which ist not recommended. What she did was aliquoting in 5ul and I have no idea why, she put 45ul water into the buffer. So in panic, my first idea was to dry the liquid in the speedvac (which took about 1hour) and to resuspend in the 5ul water again to have the original concentration. My question is whether the buffer will still be OK for cloning? I guess it is the ATP degradation that is crucial for the ligase performance - will the ATP be affected by the speedvac drying procedure? Its buffer for more than 20 samples and it would be pitty to throw it away when not necessary. I would very appreciate an advice or your experiences...Thank you :)

#2 bob1

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Posted 11 March 2013 - 12:11 PM

I think it will be degraded. It is mostly the ATP that goes off for the ligase, but the DTT can oxidise as well.




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