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Gel Purification: When is it safe to pause?

gel purification

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#1 artikisca



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Posted 03 July 2015 - 12:24 PM

I just ran my PCR product on a gel and I'm ready to excise the bands for gel purification, but the facility that houses the transilluminator has just closed for the day. Is it safe to freeze my entire gel in -20C and continue with the extraction tomorrow, or will I have to rerun the PCR? 
**Just for reference, I'm anticipating using the purified product for TA cloning-- if it is okay to freeze, will it ultimately affect the efficiency of ligation?

#2 phage434



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Posted 03 July 2015 - 01:24 PM

Freezing agarose is generally a bad idea, but this may work. The DNA will be chemically stable, but may diffuse, due to both diffusion in the agarose, and due to formation of ice crystals. I think your success will depend on how sharp a band you need. This, in turn, will depend partly on the size of the fragment -- shorter fragments will have more diffusion. At this point, you have little to lose in putting the gel in the freezer and seeing what happens. At worst, you throw it out, at best, keep going.

#3 gvbdxz


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Posted 06 July 2015 - 04:37 AM

I agree with Phage, you could also put immerse your gel in some clean buffer solution and leave it overnight at 4 degrees to avoid having to subject your gel to a freeze-thaw shock which might do funny things because of the crystallisation.

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